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ADVANCES IN FOOD RESEARCH, VOL. 28

CHEMICAL, BIOCHEMICAL, FUNCTIONAL, AND NUTRITIONAL CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

A . ASGHAR' AND R.L. HENRICKSON

Oklahoma Agricultural Experiment Station, Oklahoma State Universiry,

Stillwater, Oklahoma

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Morphology of . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A. Different Genetic Types of Collagen. . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Isolation and Identification of Collagen Types . . . . . . . . . . . . . . . . . . . .

A. Amino Acid Composition.. . . . . . . . . . . . . . , . . . . . . . , . . . . . . . . . . . . . B. Molecular Organization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. D. Functional Role of Amino Acids . . . . . . . . . . . . . . . .

F. Interaction with Carbohydrates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G . Polysaccharides of Connective Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . .

I. Metabolism of Collagen . . . . . . . . , . , . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . .

B. Catabolism of Collagen.. . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . Factors Affecting Collagen Composition and Structure . . . . . . . . . . . . . . . . . A. Antemortem Factors . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Postmortem Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A. Water Binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

111. Chemistry of Collagen . . . . . . . . . . . . . , . , . . . . . . . . . . . . . . . . . . . .

Amino Acid Sequences.. . . . . . . . . . . . . . . . . . . . . . . .

E. Type and Nature of Interchain Cross- . . . . . . . . . . . . . . . . . . . .

H. Immunochemistry of Collagen . . . . . . . . . . . . . . . . . . . . . . . . . . .

A. Biosynthesis on Polyribosomes. . . . . . . . . . . . . . . . . . . . . . . . .

Functions of Collagen in Tissues.. . . . . . . . . . . . . . . . . . . . . . IV.

V.

VI. Functional Properties of Collagen in Food Systems . . . . . . . . . .

C. Emulsifyi ty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Foaming. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. .

Swelling. .. . . . . , . , . . . .

E. Viscoelasticity . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . , . . . . . . . . . . . . .

232 233 233 238 240 24 1 245 241 250 25 1 260 26 1 266 266 261 261 212 214 215 284 287 288 306 309 311 312

'Present address: Department of Food Technology, University of Agriculture, Fasialaband, Pakistan.

23 I Copyright 0 1982 by Academic Press. Inc.

All rights of reproduction in any form reserved ISBN 0-12-016428-0

232 A. ASGHAR AND R. L. HENRICKSON

VII. Nutritional Aspects of Collagen.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312 A. Protein Quality Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313 B. Digestibility of Collage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318 C. Biological Value and PER of Collagen . . . . . . . . . . . . . . . . . . . . . . . . . . 319 D. Possible Fortification Methods of Collagen.. . . . . . . . . . . . . . . . . . . . . . 320

VIII. Food Uses of Collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322 A.

C.

Production of Edible Fibrous Collagen . . . . . . . . . . . . . . . .

Various Uses of Collagen as Gelatin

IX. Research Needs . . . . . . . . . . . . . . . . . . . . . . . . . 331 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333

I. INTRODUCTION

The unique features of connective and skeletal tissues are their diversity and morphological character. In addition, these tissues are composed basically of varying proportions of similar extracellular constituents. Advances in connective tissue research have revealed the presence of several kinds of such tissue whose organization is designed by nature to perform specific biological functions in the animal body. The functional properties of connective tissue in vivo are mainly determined by the macromolecular organization of the collagen structure, which is the principal component of connective tissue. For example, collagen constitutes over 70% of the dry weight of skin, tendon, and cartilage (Grant and Jackson, 1976), yet the physical properties of each tissue are different. The general agreement among scientists is that various macromolecular structures result from distinct genetically determined types of collagen (Harwood, 1979). Evolutionary processes probably gave rise to various types of collagen by alter- ing the amino acid sequence but preserving the general structural features to perform different biochemical functions in the body.

Apart from the physiological significance of collagen in various tissues, its bearing on the texture of meat is also well recognized. For that matter, both the quantity of collagen and its quality characteristics (extent of cross-linkages) are important (Bailey, 1972; Asghar and Yeates, 1978). From a nutritional point of view, collagen is an incomplete protein since it is limiting in some essential amino acids such as methionine, lysine, and threonine, and is practically devoid of tryptophan (McClain ef al . , 1971). However, collagen, on account of its unique structural characteristics, possesses many potential functional properties (moisturizing, binding, texturizing , lubricating, viscoelastic, emulsifying, synergistic), which it can impart under appropriate conditions in various food systems. Collagen also has many other industrial uses. These facts probably enticed Battista (1975) to state, “Nature has produced in collagen a polymer architecture

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS 233

of remarkable sophistication, an ‘engineering’ achievement that qualifies collagen for a role of far greater versatility and complexity than any other known man-made or natural high-molecular-weight polymer. ”

This article is designed to review the current information on the biology, chemistry, biochemistry, and nutritional aspects of collagen with special reference to its potential uses in various food systems.

II. MORPHOLOGY OF COLLAGEN

Morphologically, connective tissue consists of three distinct components: fibrous proteins, ground substance, and cells. The major fibrous proteins include mainly collagen with some elastin and reticulin. The ground substance occupies the extracellular space of the connective tissue as a viscous fluid derived from plasma (Fitton-Jackson, 1964), which is composed of globular mucoprotein. The proteins are associated with mucopolysaccharides, such as hyaluronic acid, chondroitin sulfates A, B, and C, keratosulfate, heparitin sulfate, and heparin in the form of galactosamine or glucosamine. The proportion of the various mucopolysaccharides in ground substance varies in different tissues. Generally two types of cell populations have been recognized in the extracellular space: fixed and wandering cells. The fixed cells comprise fibroblasts, mesenchyme cells, and adipose fat storage cells. The wandering cells comprise mast cells, macrophages or histiocytes, lymph cells, eosinophiles, and plasma cells, and are concerned mainly with controlling infection (Fitton-Jackson, 1965; Schubert and Hamerman, 1968; Forrest et al., 1975).

A. DIFFERENT GENETIC TYPES OF COLLAGEN

Before 1970, all vertebrate collagens were regarded as a simple class of molecules composed of two a1 chains and one a2 chain, with only minor heterogeneity in composition between species. During the last decade, this view has been changed by the discoveries of several genetically distinct forms of collagen having different chemical composition of a 1 chains. About 10 distinct collagen types have been reported so far with different degrees of precision, and these are believed to be the products of at least 10 nonallelic structural genes (Harwood, 1979). However, the presence of five types of a chains, namely, al(I) , al(II), aI(III), al(IV), and a2 chains, is well established in collagen molecules from various sources (Miller, 1973; Epstein, 1974; Johnson et al., 1974; Epstein and Munderloh, 1975; Slutskii and Simkhovich, 1980). These a chains constitute various types of collagen which are genetically distinct and differ in primary structure. Many researchers succeeded recently in isolating the mRNA (Diaz de Le6n et al., 1977; Monson and Goodman, 1978), and in cloning

234 A. ASGHAR AND R . L. HENRICKSON

the DNA fragments corresponding to parts of the message (Lehrach et al . , 1978; Sobel et al., 1978; Graves et al., 1979).

I . Collagen Types

The salient features of the four forms of collagen are shown in Table I. Type I collagen is composed of two identical al(1) chains and one a 2 chain and denoted as [a1(1)]2[a2(1)]. It is found in mature skin, tendon, bone, and cornea. Type I1 collagen from cartilage is composed of three identical al(I1) chains and is designated [cxl(H)13. Type I11 collagen, found in human fetal dermis and the cardiovascular system, is composed of three identical al(II1) chains and is named (al(III)l3. Type IV collagen is found in the basement membrane. This collagen is composed of three identical al(1V) chains and is designated [al(IV)],.

Some recent reports have indicated the existence of molecular heterogeneity within collagen types (Crouch et al., 1980). Tissue culture studies by Benya et al. (1977, 1978) on rabbit chondrocytes have shown that dedifferentiated chondrocytes in four subcultures also produced the type I trimer, [a1(I)I3, in addition to type I and 111 collagens, and two new pepsin-resistant collagen chains X and Y having the chain composition X,Y. They considered the new chains as the product of expression of two different collagen genes. The possible existence of additional a chains in the basement membrane of human placenta has also been indicated by Burgeson et al. (1976) and Burgeson and Hollister (1977). The new chains were designated as aA and aB, which constituted collagen type [aA(aB),]. Chung et al. (1976) have indicated the presence of additional chains, designated as A and B, in several human tissues. These chains are believed to constitute collagen [A], and [BI3 types, which closely resembled type IV collagen but lack cysteine. Two other polypeptide chains of collagen, called CP55 (Chung et al., 1976) and CP45 (Mayne et al., 1977a,b), have been identified. A study by Butler et al. (1977) has indicated that al(I1) chain in nasal cartilage is the product of more than one structural gene, which produces two types of al(I1) chains, called al(I1)-Major and cYl(I1)-Minor. They could be classified as a2(II) if they are the product of a genetic locus different from that for al(1I) chain (Bornstein and Sage, 1980). Recently, Davison et al. (1979) have also identified AB collagen from bovine cornea. They designated it as type VI on the assumption that Little and Church (1978) had already reported the presence of type V collagen in mouse embryo. Stenn et al. (1979) have also found AB, collagen.

A critical assessment of the data on the above-mentioned new chains from various sources by Bornstein and Sage (1980) suggests that A and B chains, and the recently discovered C chain (Sage and Bornstein, 1979), have many common features. They believe that there is now enough evidence to categorize XY, (Benya et al., 1977), aAaB2 (Burgeson and Hollister, 1977), and type VI (Davison et al., 1979) as collagen type V, whereas A, B, and C chains may be


designated as cil(V), a2(V) and a3(V) chain, respectively, in accordance with the accepted nomenclature. The collagen type V reported by Little and Church (1 978) may be regarded as type I trimer procollagen since it is closely related to the latter.

Somewhat divergent views have emerged from the studies which attempted to assess the chromosomal linkage of genes coding for different collagen types by following the culture hybridization technique. For instance, gene coding in the case of collagen type I has been found on chromosomes 17 (Sunder-Raj et al., 1977) and 7 (Sykes and Solomon, 1978). Recently, Kefalides (1979) has also assigned the gene coding for collagen type IV to chromosome 17. Bomstein and Sage (1980) have emphasized that collagen types, by definition, are the products of different genetic loci and are nonallelic. Hence, the term “genetic polymorphism,” which has frequently been used in the literature to describe the molecular heterogeneity of collagen (Grant and Jackson, 1976), should be avoided because genetic polymorphism generally indicates structural differences that arise in proteins coded for by different alleles at a single genetic locus. The hemoglobin variants and immunoglobulin allotypes are the examples of genetic polymorphism.

2. Collagen Types in Muscle

It appears that very few, if any, of the collagen types have a unique distribution in different tissues. More than one type seem to be present in a particular tissue. The inter- and intramuscular connective tissue (mainly collagen) has long been classified on a histological basis as the epimysium, perimysium, and endomysium (Cassens, 1971; Lawrie, 1974). Little was known about the isomorphic composition of collagen present in these layers of connective tissue until the late 1970s (Bailey and Sims, 1977; Duance et al., 1977; Wu, 1978; Bailey et al., 1979). All of the collagen types, except type 11, have been found to exist in skeletal muscle. According to Bailey and Sims (1977), type I collagen, [(~1(1)]~(~2(1), is the major component of epimysium and perimysium, whereas type IV, [ ( ~ l ( I v ) ] ~ , is confined to the endomysium. Type I11 collagen, [(~1(111)]~, was mainly identified in the perimysium and to a lesser extent in the endomysium. However, Wu’s studies ( 1978) on intramuscular bovine connective tissue indicated the presence of two different types of (Y chains in type IV collagen, namely aA(IV) and aB(IV). Both of these chains contain less alanine and glycine, and more threonine, glutamic acid, leucine, and hydroxylysine than do collagen types I and 111. The aB(1V) chain has slower electrophoretic mobility than aA(IV) chain, but both are resistant to peptic digestion. Wu (1978) has further shown that there were 5-10 mg of type 111, 10-20 mg of type IV, and 50-150 mg of type I collagen in 100 g of fresh muscle from good-grade steers. Some studies have suggested that type I11 collagen is essential for normal tensile

N W m

TABLE 1 DISTINCT FEATURES OF FOUR TYPES OF COLLAGEN AND OTHER NEWLY DISCOVERED a CHAINS IN DIFFERENT TISSUESO

Type I Type 11 Type Ill Type IV Characteristics collagen collagen collagen collagen Newly discovered a-chains

a-Chain composition Molecular formula Carbohydrate content Hydroxylation of lysineb

Hydroxylation of prolineh 1. 4-Isomer

2. 3-Isomer GI ycos ylationb

1. Hydroxylysine- galactose

2. Hydroxylysine- galactose-glucosyl galactose

2al (I) and la2(I)

10% [a~(I ) Iz [a2(~)1

0.5% in al chain 0.8% in a2 chain

-11% in a1 chain -10% in a2 chain

-0.1% in both chains

0.06% in al chain 0.15% in a2 chain 0.30% in both chains

3aI(lI)

1 wc [ a l ( I I ) I 3

2.3%

-10.0%

-0.2%

0.4%

0.5%

3a I(II1) [al(III)Is

10% 0.5%

-12.5%

~

0.01%

0.08%

3a I (IV)

1 wo 4.5-5.7%

[al(IV)l3

13%

1%

0.2%

3.2%

A “41, -

2.2%

-11%

0.7%

0.3%

0.5%

B [Bls

3.9%

-

1 wo

- I%

0.5%

2.9%

CP55 - -

4.8%

6.5%

Absent

0.7%

4.1%

Amino acidsb I . Glycine 2. Alanine 3. Cysteine

Fibril size

>33% 3 3% <33% < 10% <lWo < 10% Absent Absent Absent - - -

Occurrence

33% 3 3% >33% <33% <33% I I % 1 wa < 10% < 10% < 10%

Absent Absent Present Present Absent Relatively large and bulky Relatively Smaller than - -

delicate type I and narrow

Skin, bone, tendon, lung, Cartilage, Skin, artery, Basement Amniotic and Skin, liver, Aorta, skin, Endo- aorta, muscle ligament, cornea, uterus, membrane, chorionic epithelial liver, pla- thelial dentin, blood vessels retinal muscle muscle, membrane basement centa, base-

tissue lung, blood lens of placenta membrane smooth ment vessels, capsule muscle mem- intestine cell brane

membrane

aThese data have been derived from the sources mentioned in the footnotes of Table 11. bResiduesll00 amino acid residues.

N w -4


strength of skin and intestinal tissues (Eyre and Glimcher, 1972; Pope et al., 1975).

B. ISOLATION AND IDENTIFICATION OF COLLAGEN TYPES

Traditionally collagen has been regarded as an insoluble protein. However, Go11 (1965) has quoted Orekhovitch et al. (1948) as saying that a French re- searcher, Zachariades, showed in 1900 that collagen could be solubilized in dilute acetic acid. Later on, many different conditions were explored to solubil- ize collagen by varying pH and the salt concentrations of the extracting medium. Most of the methods fall into two categories: (1) those in which extraction was carried out at pH 3-4 with low salt concentration, the extracted product being designated as acid-soluble collagen or procollagen (Orekhovitch, 1958); and (2) those in which the extraction was performed at pH close to 7 with high salt concentration, yielding a product called neutral salt-soluble collagen or tropocollagen (Gross et al., 1955). Further characterization of these collagen extracts revealed monodispersed tropocollagen, composed of different molecular weight compounds (Orekhovitch and Shpikitar, 1955), the lighter one designated a- and the heavier ones as P-components ( P I PI2 , P.-J (Grassman etal., 1961; Piez et al., 1961, 1963).

Collagen has also been partitioned into four classes on the basis of solubility in different buffer systems (Goll, 1965): neutral salt-soluble, acid-soluble, alkali- soluble, and alkali-insoluble. The collagen, in cold neutral salt solution, represents newly synthesized molecules in a loose state, that is, a chain monomers (Jackson and Bentley, 1960). However, Gross (1964a) believes that it is the low temperature of the solution which induces solubility rather than the presence of salt, as little collagen may be extracted at body temperature. The hydrogen- bonding capacity of water increases at a low temperature and may facilitate hydration and disruption of the molecules (Kauzmann, 1964).

The acid-soluble fraction generally comprises two different aggregates besides a subunits. One, designated as P2 or P, is composed of two a1 chains, and the second, P, or P12, consists of one a1 and a2 chain linked by ester-type covalent bonds (Piez et al., 1961; Gallop, 1964). A small amount of a trimer, y-component, has also been reported in acid extract (Altgelt et al., 1961). Salt-soluble collagen was found to consist exclusively of 90% a chains and some P forms (lo%), whereas acid-soluble collagen is about 10% a chains and 90% in the P form (Piez et al. , 1965; Delaunay and Bazin, 1974). The schematic diagrams in Fig. 1 show the native and some reconstituted forms of collagen. Only 50% as many ionizable groups are present in acid-soluble as in salt-soluble collagen. The remaining groups are possibly involved in cross-linkages (Hartman and Baker- man, 1966). On the other hand, alkali-soluble and insoluble fractions represent mature collagen aggregates of higher degree.


FIG. 1 . The fibroblasts (a) produce tropocollagen molecules (b), which overlap to form native collagen (c). The newly synthesized tropocollagen is soluble in cold salt solution (d) and forms reconstituted fibrils like native collagen on warming. The native collagen can also be dispersed in acetic acid (e), which on reacting with adenosine triphosphate (ATP) produces nonoverlapping segment long-spacing (SLS) collagen (f), whereas on reacting with glycoprotein it yields fibrous long-spacing (FLS) collagen (8). From Seifter and Gallop (1966).

Different collagen types are generally isolated in the native form from various tissue by differential salt precipitation at neutral pH with NaCl (Kefalides, 1971, 1972; Trelstad et al., 1970, 1976; Chung and Miller, 1974; Burgeson et al., 1976) or (NH,),SO,, or by EtOH (Trelstad et al., 1976). For example, collagen types I11 and I precipitate in the ranges of 1.5 to 1.7 M and 2.2 to 2.5 M NaCl, respectively, whereas types I1 and IV precipitate in the range of 4.0 to 4.4 M NaCl. Further purification is performed by ion-exchange chromatography using mostly carboxymethyl cellulose and occasionally molecular sieve chromatography (Piez, 1967; Chung and Miller, 1974; Epstein, 1974). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has also been used to separate different species of collagen (Eyre and Muir, 1975a,b; Burgeson et al., 1976; Bailey and Sims, 1976; Scott and Veis, 1976; Scott et al., 1976; Fessler and Fessler, 1978; Reiser and Last, 1980). The latest methods involved differential denaturation and renaturation (Chandra-Rajan, 1978) and covalent chro-


matography on activated thiol-Sepharose (Angermann and Barrach, 1979) to separate different types of collagen.

111. CHEMISTRY OF COLLAGEN

Numerous comprehensive reviews have appeared on the chemistry and biochemistry of collagen and procollagen (Ramachandran, 1968; Kuhn, 1969; Traub and Piez, 1971; Gallop et al., 1972; Fietzek and Kuhn, 1976; Bornstein, 1974; Martin et al., 1975; Piez, 1976; Prockop et al., 1976; Bornstein and Traub, 1979). Collagen, a glycoprotein, is the longest of all protein molecules and is composed of tropocollagen monomers which are 300 nm long and 1.5 nm in diameter (Piez, 1967; Woodhead-Galloway et al., 1975; Hanvood, 1979). Each tropocollagen monomer comprises three polypeptide (Y chains, each having a molecular weight of 95,000. In view of the discovery of different types of collagen, the three a chains in a tropocollagen monomer may be identical (as in the case of collagen types 11, 111, and IV) or different (Fig. 2). An example of the latter case is type I collagen, whose monomers are composed of two a 1 chains and one 012 chain (Piez, 1966, 1967; Kuhn, 1969; Miller, 1973; Fietzek and Kuhn, 1976). Each a chain is coiled into a left-handed helix with about three amino acids per turn, but the trimers are supercoiled in a right-handed helix (Piez et al., 1963; Ramachandran and Ramakrishnan, 1976).

It will be obvious from the following discussion that the sequence and symmetry of the individual collagen molecules are well established. How collagen molecules aggregate to form the functional units (fibrils) of connective tissue has been a matter of speculation. Grassmann (1965) stated that the linear polymerization of tropocollagen monomers produces collagen fibrils which are arranged into parallel bundles (in tendon) or into a three-dimensional irregular network (in skin, cartilage, bone, and teeth) of macroscopic structures. It also appears that the organization of bundles is affected by the type of collagen present in a particular tissue (Nowack et al., 1976b; Lapikre et al., 1977).

The three-dimensional molecular arrangements of collagen have been discussed in great depth by E. J . Miller (1976). He has critically evaluated the three principal models, namely heuristic (Smith, 1968), tetragonal (Miller and Parry, 1973), and hexagonal (Katz and Li, 1974) and their modifications, presented by different scientists to account for the array of collagen molecules. According to these models, the collagen molecules are considered to be arranged in long five- stranded microfibrils with 1 0 and 4 0 axial intermolecular staggers (where D =

668 A). The microfibrils are supercoiled with a pitch of about 700 p\, and arranged face to face throughout the fibril on a tetragonal lattice of side 38.5 A. E. J. Miller (1976) concluded that there is general agreement among different

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS 24 1

A

Triple helix 1052 residues

Triple helix 1101 residues

0

C

c Disulfide linkages

FIG. 2. The helical part of the a chains is supercoiled in a right-handed helix in different types of collagen. Type I collagen consists of two identical a chains and one different a chain (A), whereas types I1 and I11 collagen are composed of three identical a chains (B and C). In the case of type I11 collagen, interchain disulfide bonds are also present within the helical region (C).

views on certain points. For instance, a collagen fibril is regarded as a single crystal in which collagen molecules are not parallel to the fibril axis, but they are tilted about 4". A vector with length 38 A is considered important in the lateral arrangement of the molecules, as is the fact that the collagen molecules, shifted axially by 40, are covalently linked in the fibril. Some aspects of the three- dimensional molecular arrangements of collagen have not been established, however, since none of the models satisfy all the necessary requirements (E. J. Miller, 1976). Although studies by Fraser ef al. (1976) support tetragonal patterns, very recently Hulmes and Miller (1979) presented evidence in favor of quasi-hexagonal packings.

A. AMINO ACID COMPOSITION

Twenty or twenty-one different amino acids are known to be present in different collagen types. The overall amino acid composition of mammalian collagen type I is shown in Fig. 3. The amino acid composition of collagen is unique in some respects among other proteins. For example, it is extraordinarily rich in glycine and proline, and contains large amounts of hydroxyproline, whereas tryptophan is absent. Cysteine is present only in collagen types 111, IV, and CP55, and methionine is the only sulfur-containing amino acid in collagen types I and 11. It can be seen that 33% of the total amino acid residues consist of


FIG. 3. acidic, hydroxy, polar, and nonpolar amino acids of type I collagen, [oll(I)]2[2a].

Diagrammatic representation of the amino acid composition and relative proportions of

glycine, about 12% of proline, 11% each of alanine and hydroxyproline, total imino acid residues being about 23%. Relatively small amounts of each of the other 14 amino acids account for the remaining proportion of the residues. The content of hydroxylysine, histidine, phenylalanine, isoleucine, tyrosine, and sulfur-containing amino acids is about 1% or less for each. Polar amino acid residues constitute about 40% of the molecule, of which 11% are basic and 9% acidic amino acids and about 17% are hydroxy amino acids. About 5% of the aspartic and glutamic acid residues are present as amides. There are approximately four asparagines for every glutamine in collagen (Cassel and McKenna, 1953). About 60% of the molecule consists of nonpolar (hydrophobic) amino acid residues.

Table I1 presents the amino acid composition of different isoformic collagen ci chains. It shows that major variations occur in the content of 3- or 4-hydroxyproline, glutamic acid, proline, valine, isoleucine, leucine, hydroxylysine, lysine, and histidine in different types of a chains, and hence in collagen. Though hydroxyproline and hydroxylysine are considered to be specific to collagen, these residues have been found in the complement component C lq (Reid, 1974) and in elastin (Gosline, 1976; Sandberg, 1976; Franzblau, 1971).

TABLE I1 AMINO ACID COMPOSITION OF DIFFERENT a CHAINS OF COLLAGEN ISOLATED FROM VARIOUS TISSUES

Residuesilo0 amino acid residues

3-Hydroxyproline 4-Hydrox yproline Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Half-cystine Methionine Isoleucine Leucine

1.1 114 46 18 35 77

118 330 1 I9 19

ND 5.3 8.2

20

1.2 105 45 18 30 70

114 33 1 105 35

ND

16 33

4.7

2.0 99 42 20 27 89

121 333 100 18

ND 9 9

26

1 .o 97 50 23 28 93

113 336 102 23

ND 5.7 8.8

26

ND ND' 11 2.5 121 125 130 109 48 42 51 50 15 13 23 26 41 39 37 31 71 71 84 84

102 107 61 97 355 350 310 319 92 96 33 52 16 14 29 27 2 2 8 ND 7 8 10 1 1

13 13 30 16 21 22 54 35

2.9 7 10 0 109 113 105 65 50 49 49 78 19 29 21 16 26 34 25 27 91 86 95 104

118 98 120 92 322 346 334 318 46 54 45 41 18 28 22 21

ND 0 0 20 8 11 9 8

19 12 15 20 39 33 38 24

(continued)

TABLE I1 (Conrinued)

Residues/ I00 amino acid residues

Tyros i n e Phenylalanine Hydroxylysine Lysine Histidine Arginine 3- + 4-OH-Proline Proline + 3- + 4-OH-Proline OH-Lysine Lysine + OH-Lysine

2.1 13 10 27 4

49 0.49

0.27

3.2 11 12 20 10 51 0.48

0.38

1 13 14 22 2

51 0.46

0.39

1.1 12 18 17

43 2.4

0.46

0.51

2 3 6 1.8 8 8 27 14 5 5 44 24

30 30 10 18 6 6 10 11

46 46 33 68 0.54 0.54 0.70 0.54

0.14 0.14 0.81 0.57

2.1 12 35 20

50 7.5

0.49

0.64

0 10 22 12 8

48 0.55

0.65

3 18 11 15 39 48 13 18 6 3

40 64 0.49 0.41

0.75 0.73

OHuman placental membrane type 1 (Y chain composition from Burgeson et al. (1976) bHuman articular cartilage type I1 composition calculated from Miller and Lunde (1973). cLathyritic chick xiphoid cartilage type I1 collagen composition from Trelstad et al. (1972). dHuman skin type 111 collagen Composition from Epstein (1974). eHuman dermis, aorta, and uterine leiomyoma type 111 composition from Chung and Miller (1974). fHuman glomerular basement membrane type IV composition from Kefalides (1971). gChains isolated from human placental membranes [from Burgeson et a/. (1976)l. hAmino acid composition of chains isolated by Chung e t a / . (1976). 'ND, Not determined.


B. MOLECULAR ORGANIZATION

The synthesis of a protein in living cells involves placing the amino acids in the proper sequence, determined by the genetic code, and linking them together by a peptide linkages. The polypeptide chains of the native proteins, however, are not freely flexible (random coils) due to certain restrictions imposed by the requirement that bond angles be maintained close to certain fixed degrees. The peptide bond is stabilized by delocalization of n-electrons of the C=O bond into the C-N bond. This imposes restrictions on the rotation about the C-N bond; however, side groups of amino acids may freely rotate around the a-carbon. The resonance hybrid possesses about 40% double-bond character in the C-N bond, whereas 60% double-bond character remains with C = O bond (Cram and Cram, 1978). The planar geometry of the peptide linkage is as follows:

However, the primary structure of collagen has some unusual features. For instance, some degree of nonplanar distortion in collagen structure is now considered possible to account for its increased stability. The peptide linkages in other proteins invariably have a trans configuration, whereas in the collagen structure a cis configuration is also possible at those places where proline and hydroxyproline are involved in the peptide linkages. As the free-energy difference in the two geometrical isomers is insignificant, either one of the stereo forms of peptide bond can occur to suit the requirement of the structure (Badger and Pullin, 1954; Wyckoff et al., 1970; Ramachandran and Ramakrishnan, 1976).

@ and 9, respectively, denote the dihedral angles of rotation between C-N and C--C bonds. Since the a-carbon of proline and hydroxyproline is covalently linked to the N atom to form a five-membered ring, the dihedral angle r$ about the C - C bond can only be close to -60" (Ramachandran and Ramakrishnan, 1976).

Another unusual feature of the collagen structure was once thought to be the presence of y-glutamyl peptide linkage (Gallop et al., 1960), however, in some natural peptides, glutamic acid has been found to occur in such linkages (Hard- ing, 1965; Seifter and Gallop, 1966). Franzblau et al. (1963, 1970) have re-


ported that about 40% of the COOH groups of glutamic acid in the primary chain of collagen are involved in peptide linkages through the y-COOH group. The amorphous regions (polar) of the a chains mainly contain this type of intramolecular bond (Rojkind and Gallop, 1963) and presumably serve to disrupt the helical structure imposed by the crystalline regions, hence providing regions of flexible, loose conformation at specific intervals along the collagen fibrils (Seif- ter et al., 1965; Gallop et al., 1965, 1967).

A chemical method was used to determine how much of y-COOH groups of glutamic acid residues are free or bound in y-glutamyl linkage. The groups were converted into hydroxamic acid by reacting with carbodiimide and hydroxylamine followed by dinitrophenylation, Lossen reaction, and acid hydrolysis (Franzblau et al., 1963). However, the reaction was not precisely specific since some amide groups were also affected. Hall (1976) suspected such isopeptide linkages (y-glutamyl) were artifacts brought about during hydrolysis of collagen in the preparatory steps. The use of a sequence of proteases and peptidases, specific for a peptide bond, which would hydrolyze a protein completely to its amino acids, would be a more suitable approach to ascertain the exact nature of the bonds. By following such an approach, Bensusan (1969) successfully released all the glutamic acid residues in collagen by using a mixture of proteolytic enzymes which did not cleave model peptides containing y-glutamyl bonds. Hence this study did not support the presence of y-glutamyl bonds in collagen.

At one time the presence of ester bonds was also regarded as one of the special features of the collagen fibril. The first experimental evidence was based on the use of such nucleophilic reagents as hydroxylamine or hydrazine for cleaving the probable ester bonds under defined conditions of pH and temperature (Gallop et al., 1959). Since the cleavage of some special imide bonds was also possible under those conditions, the term “ester-like’’ bonds thereafter has been used to describe them (Gallop, 1964). It was proposed that the four subunits of a chain (Petruska and Hodge, 1964) are linked by three pairs of ester-like bonds, and that the two aspartic acid residues in the C-terminal region of the a chain provide the paired acyl donors for ester-like intrachain bonding (Blumenfeld and Gallop, 1962). One of the esters involves the a-COOH of one aspartyl residue, and the other involves the (3-COOH group of the second aspartyl residues (Seifter et al., 1965). It was also ascertained that glutamyl residues were not acyl donors for ester-like bonds. Although the nature of the alcohol function (if an ester is present) or amide function (if a special imide is present) has never been determined. Seifter. and Gallop (1966) proposed the possibility that the “bound masked aldehyde” that occurs in collagen, represents ester formation between aspartic acid residues and hemiacetal or vinyl alcohol functions. Contrary to this, the present consensus does not support the presence of ester-like linkages in collagen (Hall, 1976; Bornstein and Traub, 1979).


C. AMINO ACID SEQUENCES

Presently, the complete sequence of the 1052 amino acid residues in the al(1) chain is known (Hulmes et al., 1973; Fietzek and Kiihn, 1976) and about two- thirds of the amino acid sequence of the a2(I) chain has also been established (Fietzek and Kiihn, 1976). Some data on peptides, derived by hydrolyzing with cyanogen bromide from other collagen types, are also available (Dixit et al., 1975; E. J . Miller, 1976; Fietzek et al., 1977). The latest reports, together with the earlier findings, suggest that 50-60% of an a chain consists of nonpolar sequences, which are expressed as tripeptides with the general formula Gly-X-Y (where X is generally proline and Y is mostly hydroxyproline). About two-thirds of the X and Y positions are occupied by a variety of other amino acids which, although they decrease the stability of the triple helical, are essential for the organization of collagen at the fibrils level. These amino acids tend to be clus- tered in groups of hydrophilic and hydrophobic residues. The precise sequence of amino acids in the X and Y positions in various a chains is different. These sequences represent the crystalline regions. Figure 4 presents the details of such a sequence schematically for a tropocollagen molecule. However, the original proposal of Petruska and Hodge (1964), that a chains of tropocollagen are built from smaller subunits which are joined approximately end-to-end by ester-like bonds (Fig. 4), is no longer considered valid (Traub and Piez, 1971).

As mentioned earlier, each tropocollagen unit consists of three polypeptide strands of a chains. Each chain contains alternative amorphous and crystalline regions (Bear, 1952). When viewed under an electron microscope, the regions composed mainly of the nonpolar amino acid residues, and representing crystalline zones, appear light (Fig. 4). The amorphous regions (pyrrolidine- poor), containing ionic polar amino acids which can take up stain, appear as dark bands (Grassman et al., 1956; Kiihn, 1960; Hanning and Nordwig, 1965; Chap- man and Hardcastle, 1974). It may be pointed out that the intrachain subunits proposition of Petruska and Hodge (1964) requires that the a chains have different repeating distributions of ionic groups. However, the studies by Tkocz and Kiihn (1969) suggest that a 1 and a 2 chains must contain a similar distribution of ionic groups. There is no difference of opinion that the tropocollagen fibrils are arranged relative to adjacent molecules so as to allow for staggered overlap of one another by about three-quarters of their length (Fig. I). The cross-linkages (hydrogen and covalent bonds) bind the tropocollagen molecules to their laterally placed neighbors (Bruns and Gross, 1973; Bruns et al., 1973).

There is a sequence of 24 amino acids with basic and acidic residues in the amorphous regions. The basic parts contain two hydroxylysine, three arginine, and one histidine residues (but no proline or hydroxyproline) in the a1 chain (Chapman, 1974). The acidic part of the amorphous region contains a sequence

'SEGMENT LONG SPACING' (SLSI FORM -. . -

I< .. . >\ I

- - _ _ -.

.-. . .

y - h - t w o r ~ ~ ~ ~ w + G I ~ - P ~ ~ - < G ~y -Pro 4 n.1

DIALYZABLE PEPTIDES ,' NON DIALYZABLE RAPIDLY

PEPTIDES DIALYZABLE PEPTIDES STALLINE' '*AMORPHOUS' "CRYSTALLINE' "AMORPHO~S"

(BAND) lNTLRB,AND (BAND)

/ \ / i

\

ly-Pro (Gly, GluZ Asp, Lysi AloZ SwlX f Pro

(Gljs, Alol. Serq.Glu1l IGlyj. Alai. %r,. Valtl

lG ly j .Abq. Val,. Thrll

FIG. 4 Little, Brown and Company, Boston

Schematic presentation of the collagen fibnl packing, showing the sequence of subunit structure From Gallop (1964) Courtesy of


Non helical segment Collagen-like segment Globular segment

Nonhelical Triple helical domain Nonhelical COOH-terminal NHJerminal domain domain

FIG. 5 . Schematic representation of type I tropocollagen monomer, composed of two identical pro-a1 chains (solid lines) and one pro-a2 chain (dashed line). The central part of the molecule is triple helical. The nonhelical COOH-terminal domain contains the interchain disulfide bonds, whereas the nonhelical NH2-terminal region is composed of a presumably globular segment, a short collagen-like segment, and a nonhelical segment where cleavage by amino-terminal protease occurs.

of 16 amino acids with only one imino acid, two lysine, two aspartic, and one glutamic residues. This helical region seems important for generating interchain cross-linkages. These observations seem to be at variance with earlier ideas regarding collagen structure (Astbury, 1940; Pauling and Corey, 195 1).

Earlier information on collagen structure also gave the impression that the helicity extends throughout the molecule. However, recent advances have revealed two structurally and functionally distinct regions in each a chain: a central triple helical region composed of 101 1 amino acid residues, and the N- and C- terminal nonhelical regions (Fig. 5 ) composed of 9-25 residues (Kuhn, 1969). The sequence of amino acids in the helical and nonhelical regions has been determined for collagen [al(I)],[a2(1)] from calf skin (Hulmes et al., 1973; Fietzek and Kuhn, 1976).

1 . Triple-Helical Regions

The triple-helical regions are composed of chains of tripeptide units of the general formula (Gly-X-Y), in all types of collagen. However, the distribution of amino acids between the X and Y positions is uneven (Fietzek and Kuhn, 1976). In the case of al(1) chains, glutamic acid and such hydrophobic amino acids as leucine and phenylalanine generally occupy the X position, whereas threonine and arginine are present in the Y position. Other hydrophobic amino acid residues (valine and isoleucine) are distributed randomly, whereas hydrophilic amino acids (aspartic acid and lysine) are almost evenly distributed between X and Y positions. The same seems to be true for the a 2 chain, except that valine is mainly present in the Y position, whereas threonine is evenly distributed.

Generally hydroxylation of proline at C-4 and of lysine at C-5 occur when


these amino acid residues are at the Y position, and hydroxylation of 3-hydroxyproline occurs when proline is at the X position in both of the a chains. While 54% of the lysine in the a 2 chain is hydroxylated, only 5-8% is hydroxylated in the a 1 chain, generally at the Y position (Fietzek and Kuhn, 1976). The hydrophobic amino acid residues (valine, leucine, isoleucine, methionine, phenylalanine, and tyrosine) are relatively more abundant in the a 2 chain than in the al(1) chain and they are evenly distributed (Tkocz and Kuhn, 1969).

2. Nonhelical Regions

The physicochemical evidence provided by Boedtker and Doty (1956) suggested that collagen molecules have dangling chain peptide appendages at one or both ends which are not in triple-helical conformation. These regions are also referred to as telopeptides by Rubin et al. (1963) and are devoid of hydroxyproline. Sixteen amino acid residues with the same sequence have been found in the N-terminal telopeptide of the a 1 chain of type I collagen from different species. The one variation is leucine, which occupies the N-2 position instead of methionine, in the case of calf skin collagen. Generally the N-terminal region is high in hydrophobic amino acids. Another common feature is the presence of a lysine residue at the N-8 position (Fietzek and Kuhn, 1976), which may be oxidized to an aldehyde derivative by lysine oxidases for generating intra- and intermolecular bonds (Gallop et al., 1972; Robins et al., 1973).

In contrast to the al chain, the number of amino acids in the N-terminal end of the a 2 chain varies significantly from 9 to 11 among different species. Lysine may be present at the 5th, 6th, or 7th position. Despite these differences, both the a 1 and a 2 chains begin with glutamic acid as the N-terminal amino acid (Kang and Gross, 1970; Rauterberg et al., 1972a; Fietzek et al., 1974b; Becker et al., 1975b).

The investigations on the nonhelical C-terminal end of the al(1) chain of collagen from different species have shown the presence of 25 amino acids, of which hydrophobic amino acids account for the major proportion. This region is longer than the N-telopeptide region. An oxidizable lysine residue was found at the C-16 position in the case of rabbit skin collagen and at the C-17 position in that of calf skin collagen (Rauterberg et al., 1972b; Becker et al., 1975b).

D. FUNCTIONAL ROLE OF AMINO ACIDS

The peptide linkage formed by a-NH, and a-COOH of different amino acids (other than proline and hydroxyproline) contains the NH group, which can participate in hydrogen bonding, and hence contributes to the stability of the helix of proteins. However, proline and hydroxyproline serve different purposes in the collagen structure. Walton (l974), in reviewing the collagen function in tissues,


emphasized the role of imino acid residues in the a 1 chain sequence in determining the pitch of the collagen superhelix. It has been mentioned in the previous section that the N atom of the proline and hydroxyproline residues is linked with a-C to form a rigid five-membered ring structure, hence there is no freedom of rotation about the N-C bond (the dihedral angle c$ = -60"). According to Schimmel and Flory (1968), the pyrrolidine rings impose conformational re- straints on residues preceding but not following them in the peptide chain sequence. Besides, as there is no H atom in imino peptide linkage, it cannot participate in hydrogen bond formation. These characteristics of imino acids are important in disruption of the helicity of polypeptide chains.

On the other hand, hydroxyproline plays a part in the stability of collagen's minor and superhelix by hydrogen bonding, which involve the oxygen of hydroxyproline's hydroxyl group with the backbone of the collagen triple helix via a water dipole (von Hippel, 1967; Ramachandran and Ramakrishnan, 1976). The ability of the a chains to attain the triple-helical conformation and its thermal stability depends not only on the content of the proline and hydroxyproline residues (Gustavson, 1955; Josse and Harrington, 1964; Sakakibra et al. , 1973; Berg and Prockop, 1973; Jimenez et al., 1973; Jimenez and Yankowski, 1975), but also on the distribution of these residues along the chains (Berg and Prockop, 1973). Segal (1969) has shown more specifically that with proline-containing tripeptides, the stability of the helix falls in the following order: Gly-Pro-Pro > Gly-Pro-Y > Gly-X-Pro > Gly-X-Y. Regarding the role of hydroxyproline, Sakakibra et al. (1973) have indicated that it imparts exceptional stability when present in the Y position. Generally, collagen type [al(I)],[a2(1)] and [al(I)I3 are more stable than type [a2], (Tkocz and Kuhn, 1969).

The distribution of polar and hydrophobic amino acid residues determines the ordered aggregation of molecules into fibrils (Highberger et al., 1971; Fietzek et al., 1974a). The acidic and basic amino acid residues generally occur in close proximity to each other, concentrated more in some regions than others; hence they are important in interchain cross-linking with adjacent molecules in the formation of collagen fibrils (Tkocz and Kuhn, 1969). Unlike hydroxyproline, which is found only in the helical regions of the molecule, hydroxylysine may occur in both the helical and nonhelical N-terminal region, where it plays an important role in intermolecular cross-linkings (Tanzer, 1973; Bailey et al., 1973, 1974).

E. TYPE AND NATURE OF INTERCHAIN CROSS-LINKAGES

The early studies indicated the presence of different types of interchain cross- linkings in collagen. Despite the fact that tyrosine content is quite low in collagen, some reports have assigned a special role to tyrosine in the aggregation of soluble collagen (Bensusan and Hoyt, 1958; Bensusan and Scanu, 1960; Hodge


et al., 1960). Deasy (1962) reported the presence of peroxide cross-links (-U-O-) in collagen. It was assumed that the phenolic group of two tyrosine residues of adjacent chains oxidized to form a di-p-(2-amino-2-carbox- yethy1)phenyl peroxide linkage. LaBella and Paul (1965) supported the existence of such cross-links in collagen, but Sinex (1968) disagreed with their presence. Joseph and Bose (1962) indicated that about 30% of the guanidinyl group of arginine residues in collagen are cross-linked to the a-COOH group of glutamic acid residues. They have further shown that guanidinyl-carboxyl cross-linkages increase with the age of the rat (Joseph and Bose, 1962). Veis and Schlueter (1963) speculated on an important role for serine and threonine residues in hard collagenous tissue (dentin), where they may be involved in PO,-mediated (ester) cross-linkages. Some reports have also indicated that about 30-40% of the E-

NH, group residues form e-lysyl peptide linkage to provide branching points between chains (Mechanic and Levy, 1959; Joseph and Bose, 1962).

It appears that the evidence provided in favor of all these postulated cross- linkages has been suggestive rather than conclusive. A critical evaluation of the experimental evidence regarding different chemical bonds in collagen led Hard- ing (1965) to conclude that y-glutamyl peptide bonds exist in the primary chain, but that none are present in cross-linking between collagen chains. On the other hand, P-aspartyl peptide bonds do not exist in any significant amounts, whereas the existence of interchain elysyl peptide bonds is improbable. During the past decade much new information on this aspect has become available, thereby changing some of the previous concepts dramatically. According to prevailing views, the involvement of aromatic side chains by tyrosine and phenylalanine has not been supported (Hall, 1976) and the ester-like bonds have not been proved in type I collagen (Bornstein and Traub, 1979). Three general groups of cross-linkages in collagen have been defined. First, those linkages which fix the overlap of the ends of a chains are called head-to-tail bonds; second, those reducible cross-linkages which stabilize the aggregation of these chains are denoted as side-to-side bonds; and the third group is end-to-end bonds (Kuhn, 1969; Zimmermann et al., 1970). These cross-linkages originate by different modes of action.

The following types of bonds have been precisely defined in collagen by modern methodologies.

I . Hydrogen Bond

It is known that hydrogen linked covalently to an electronegative donor (e.g., N, 0) can form a second weak bond with another electronegative atom (acceptor). The latter is called a hydrogen bond. The common types of hydrogen bond in proteins are those between NH.-.N, NH...O, and OH...O atoms of amino acids, and they have an energy content of 3-5 kcal/mole (Ramachandran, 1968).

CHARACTERISTICS OF COLLAGEN IN FOOD SYSTEMS

(chain A)

b' H O 0 I II II

-C-C-N-C-C-N-C-C-N-C- I I 8 .. H Y

far away 4'. . I

for H bond i 0 !I

-C-C-N-C-C-N-C-C-N-C- I II (chain B) 'H 0

II I I 0 H i

(a) One-bonded s t ructure

/ I 0 H O

I II II - C- C- N-C-C-N- C-C-N-C-

z I I

H H I I

II I II -C-C-N-C-C-N-C-C-N-(

I II I H

(b) Water-bridged s t ructure J H Pol\

'.o z

H O I II II

-C- C-N- C- C-N- C-C- N- C- II I I I

c, ,c B C

'0-H ' -,\O-H 4' -. Y0\H. /

H 0 II

*O It I

-C-C-N-C-C-N-C- C-N- C- I II I

H

(chain A)

(chain B)

(chain A)

(chain B)

25 3

(c) Water-bridged s t ructure with hydroxyproline

FIG. 6. The nature of hydrogen bonds between two adjacent chains (A and B) of the collagen fibril. The structure is actually three-dimensional; the one-dimensional representation shown above with parallel chains is a simplification. After Ramachandran and Ramakrishnan (1976).


These bonds are important in stabilizing the secondary structure and packing of collagen molecules (Harrington, 1964); hence they fix the shape of the protein molecule in a specific conformation. In native collagen, the tropocollagen chains are oriented so that the NH group of the third peptide linkage of one chain may form a hydrogen bond with the COOH group of the third peptide linkage of an adjacent chain. It should be emphasized that imino peptide (involving pyrrolidine) bonds, lacking one hydrogen atom, cannot form intramolecularly hydrogen-bond stabilized (Y helical structures (Veis, 1964), although an interchain hydrogen bond can be formed between the OH group of an hydroxyproline residue on one peptide chain and the COOH group on an adjacent chain (Gustav- son, 1956). However, opinions differ as to the number of hydrogen bonds in collagen. Rich and Crick (1961) argued in favor of a “one-bonded structure,” that is, only one hydrogen bond for every three residues (Traub, 1969), whereas Ramachandran et al. (1962) believed in a “two-bonded structure,” that is, two hydrogen bonds for every three residues. Figure 6 depicts the “one-bonded’’ and “two-bonded’ ’ structures.

Ramachandran and Chandrasekaran (1968) offer an alternative “two-bonded’’ structure which reconciles the two views by indicating that one hydrogen bond is directly between adjacent polypeptide chains and the other hydrogen bond forms via a water molecule. In the revised “two-bonded’’ structure, the OH group of hydroxyproline has been assumed to perform two functions: it forms a hydrogen bond with the bridging water molecule to increase the stability of a triple-chain protofibril, and another hydrogen bond (cross-link) between one triple-helical chain and a neighboring triple-helical chain (Fig. 6C). The “two-bonded’’ structure seems to conform better to the experimental evidence (Harrington, 1964; Berendsen, 1972; Yee et al., 1974; Suzuki et al., 1980). It has been further shown that the 4-OH group of hydroxyproline in the Y position of (Gly-X-Y), is in the trans configuration with respect to the COOH group of hydroxyproline so as to perform these functions (Schubert and Hamerman, 1968; Ramachandran and Ramakrishnan, 1976).

Salem and Traub (1975) have also suggested the involvement of glutamine in the formation of hydrogen bridges in the helical region of the cx chains. Accord- ing to them, glutamine at the Y position in the [Gly-X-Y], tripeptide chain of helical regions may form a hydrogen bridge with the carbonyl group of the peptide bond (the aspargine side chain is too short for this reaction).

2. Hydrophobic Bonds

Although the nonpolar amino acids glycine and alanine constitute nearly 44% of the collagen molecule, yet they may contribute little to the nonpolar van der Waals interactions (hydrophobic bonding) due to their small side groups (Veis, 1964). Contrary to this, some studies have indicated possible hydrophobic in-


teraction between adjacent propyl residues on different chains (Segal, 1969; Yonath and Traub, 1969). However, the side group of other nonpolar amino acids may form inter- and intramolecular hydrophobic bonds (Schnell, 1968; Heidemann and Hill, 1969) in the nonpolar segments (interband regions) of a chains (Schubert and Hamerman, 1968). It is now known that the a 2 chain contains more hydrophobic amino acid residues than the a 1 chain. Thus, the former chain contributes considerably to the hydrophobic character of type I collagen (Fietzek and Kuhn, 1976). Moreover, maximal interaction of the polar and nonpolar regions of adjacent molecules is facilitated by an axial stagger (D =

233 amino acid residues) within the fibrils. Thus, both electrostatic forces and hydrophobic interactions are important in aggregation of collagen molecules into fibrils.

3. Ionic Bonds

Salt (ionic) linkages have been considered relatively unimportant in stabilizing the collagen structure as compared to hydrogen bonding (Weir and Carter, 1950), but Weinstock et al. (1967) believe that most of the ionic sites form inter- and intramolecular salt linkages. For instance, Salem and Traub (1975) have suggested that arginine at the Y position can be involved in an electrostatic interaction with glutamic acid in the X position of an adjacent chain.

4 . Covalent Bonds

a . Disulfide Linkages. Interchain disulfide (-S-S-) bonds have been found in the C-terminal extraglobular peptide region (called the P,-a chain) of procollagen a chains of all types. But, the P, chains are enzymatically cleaved before the procollagen molecule is secreted into the extracellular space (Martin et al . , 1975; Bomstein and Traub, 1979). Consequently, disulfide bonds are not present in collagen types I and I1 due to the absence of cysteine residues in their tropocollagen chains. However, interchain disulfide bonds have been reported in the helical region of type 111 collagen (Harwood, 1979) and in the glycoprotein extensions (terminal regions) of type IV collagen (Kefalides, 1973). Both of these collagens contain appreciable amounts of cysteine residues.

Cross-Linkages Involving Lysine and Hydroxylysine. It is now well documented that covalent interchain (intermolecular) cross-linkages in different collagen types originate from the reaction of aldehydes, derived from oxidative deamination of the E-NH, group of lysine and hydroxylysine residues (Tanzer, 1973, 1976; Bailey, 1969, 1974; Gallop and Paz, 1975). The lysine and hydroxylysine residues in both terminal regions of the aI(1) chain and in the N-terminal region of the a 2 chain may be oxidized to a-aminoadipic acid a-semialdehyde (Traub and Piez, 1971; Stark et a l . , 1971a; Gallop et a l . , 1972). This occurs by

b.


oxidative deamination of the E-NH, group of lysine and hydroxylysine residues by a copper ion-dependent lysine oxidase (Siegel, 1974, 1979). The resultant carbonyl compound then reacts with the E-NH, group of lysine or hydroxy- or glycosylated hydroxylysine present in adjacent molecules. The intermolecular cross-links are formed by a series of aldimine or ketoimine (Schiff base) and aldol condensation reactions, leading to the formation of highly stable pyridinium compounds such as desmosine and isodesmosine (Gallop et al. 1972; Robins et al., 1973; Bailey et al., 1974; Tanzer, 1976); their amount increases with the age of the animal.

These cross-linkages have been identified following a mild reductive reaction of collagen fibrils with borohydride, which stabilizes the bonds and makes the isolation of linked amino acids possible. However, Bailey et al. (1974) expressed the feeling that the identification of these compounds from borohydride- treated collagen may not necessarily provide proof that their nonreduced forms function as intermolecular bonds in vivo, because the initial condensation reaction might have been favored by the alkaline-reducing conditions.

Based on the known sequence of amino acids in the al(1) chain, it is now proposed that the C-terminal lysine may react with hydroxylysine at position 87 and with lysine at position 327, whereas the N-terminal amino group forms a linkage only with hydroxylysine at position 927. One side-to-side bond can only result from the reactions of the C-terminus with the regions at 327 and 564 (Kang, 1972; Miller, 1971b; Dixit and Bensusan, 1973; Becker et al., 1975a). The following are some of the important reactions which are believed to proceed from the condensation of carbonyl derivative (a-aminoadipic acid 6-semialdehyde) with other functional groups of amino acids through a series of complex mechanisms (Figs. 7 and 8).

1. Hydroxylysinonorleucine: These linkages can be developed by two reactions: first, by condensation of hydroxylysine and a-aminoadipic acid &semialdehyde; second, by condensation of lysine and 6-hydroxy a-aminoadipic acid a-semialdehyde. Such linkages were first identified by Bailey and Peach (1968) in collagen from calf and rat tendons. Later on, many studies substantiated these findings in skin (Franzblau et al., 1970; Tanzer et al., 1970; Bensusan, 1972; Bailey and Lapiere, 1973). In the case of calf skin, hydroxylysine and a-aminoadipic acid &semialdehyde are mainly involved in the cross-linkage (Tanzer and Mechanic, 1970; Nicholls and Bailey, 1980).

2. Lysinonorleucine: Lysinonorleucine links have been found in skin collagen from different species (Bailey, 1970; Kang et al., 1970; Mechanic and Tan- zer, 1970; Bensusan, 1972), tendon (Shimokomaki et al., 1972; Cannon and Davison, 1973), and basement membrane (Tanzer and Kefalides, 1973).

3. Dihydroxylysinonorleucine: The occurrence of these cross-linkages in collagen from bone and dentin was suggested by Bailey et al. (1969); Davis and Bailey (1971) identified the actual structure. Later studies reported the pres-


H I --N H C=O

‘C’

4, Cu2+ lysyl oxidase N y2

- ( p ,

FH2

- N / h = O CHO -N+c=o

N II CH + lysine I

J

J

I1 CH H I

I -N,H,c=o YH(OH) + hydroxy- 7

(FHz)* - lysine (FH,)s

H I a- Aminoadipic acid- H I Dehydrohydroxy- 6-semialdehyde (allysine) Dehydroly- lysinonorleucine sinonorleucine + allysine

nuc leophilic condensation

H I C

1 I

-N,H,C=O

(CH,),

H H -N\H,C=O C -N,E,C =O

I (CH,),

I $H -t hydroxy- CH2 +lys ine CH

(CH,),

I H C = O

“H I

( F H A I I O=C,H c-cH,-NH-(cH,),-c’ II

,C-(CH,),-CH-NH-CH,-C lysine CH-CHO HN I I I I

I (CH,),

(FHJ , I I OH

- N/g\C =O - N/$C=O -N’$\C=O H I H I H I

Hydroxymerodesmosine Aldol Merodesmosine

J + histidine

H I -N,E,C=O

1 H I

C I I

H I -“,H,C=O

C

(CH,), (CH,), I I I

--N,H,C=O

i I CH

I1 H C = O C-CH=N- (CH,),-C<

I NH

O=C\H I CH It y 2

,C- (CH,),- CH-N=CH-C C=CH-N

( + A 1;;1 (FHJ , I HN I I

I OH y. - E/$c=o - N+C=O CH2 -N’$\C=O

I 1 H I -N’&.c=o I H I

Dehydro- Aldol histidine merodesmosine

Dehydrohydroxy- merodesmosine

FIG. 7. Cross-linking reactions involving lysine derivative as intermediates

ence of such cross-linkages in skin and tendon collagen (Mechanic et al., 1971; Bensusan, 1972; Eyre and Glimcher, 1972; Forrest et al . , 1972; Bailey and Lapikre, 1973).

4. Aldol histidine: The presence of aldol histidine cross-linkages has been suggested in bovine skin and basement membrane but not in other collagens (Fainveather et al., 1972; Tanzer et al. , 1973b). This type of unreduced


H I

F - N,H,C = 0

(CH,),

7% C H I

--N,H,c=o CH(OH)

I

I

CH(OH) I

CH II

H I ‘C’ ( y J *

--N H C=O NH, Hydroxylysine residue

‘ CH(OH) H c,

I CHO I -E+c=o I -N’H c=o

Dehydrohydroxyly- sinonorleucine

Amadori rearrangement 1

Lysino-5- ketonorleucine

Dehydrodihydroxyly- sinonorleucine

6 -Hydroxy , a -aminoadipic

(hydroxyallys ine) acid- 6 - semialdehyde

I 4 Amadori rearrangement I + allysine

H I H I -N,H c = O --N H c=o

F’ ‘C’

Hydroxyaldol I + histidine

Hydroxylysino- 5- ketonorleucine

H I -N,H C=O

C’ I

CH(OH) I

(p

Hydroxyaldol histidine

FIG. 8. Cross-linking reactions involving hydroxylysine derivative as intermediates.


linkage is quite labile in acid (Kang et al., 1970; Davison et al., 1972; Robins and Bailey, 1973b, 1975).

5 . Hydroxyaldol histidine: Housley et al. (1975) have reported the presence of hydroxyaldol histidine linkages in collagen which are not reducible and are stable. They involve condensation of allysine, hydroxylysine, and histidine (Tanzer et al., 1973b).

6. Hydroxypyridiniurn: Very recently, Eyre and Oguchi (1980) have identified hydroxypyridinium cross-links in skeletal collagen. The formation of these cross-links involves two aldehyde intermediates, formed from hydroxylysine residues.

Among all these compounds only hydroxylysinonorleucine and dihydroxylysinonorleucine have been isolated from collagen in significant amounts. Their content in collagen seems to be related with the extent of lysine hydroxylation in the N- and C-terminal regions of the ci chains (Balazs, 1977). In fact, the cross- linkages, originated from lysine and hydroxylysine, are distributed in varying amounts in collagen of different tissue and species (Hall, 1976). Some tissues contain only one type of cross-link, some two, and others may contain more depending upon the ci chain’s composition (Bailey and Robins, 1976). An explanation of these observations could be that the degree of hydroxylation of the lysine residues and the proximity of the reacting groups in the terminal telopeptide of different a chains may determine the nature of the cross-linkages.

It seems that aldehydes, formed by lysyl oxidase, are involved in two main types of cross-linkages. First, intramolecular linkage may be formed to join a chains by aldol condensation. Second, intermolecular cross-linkages are formed mainly by Schiff base reaction between an aldehyde derived from lysine, hydroxylysine, or glycosylated hydroxylysine and the E-NH, group of another lysine, hydroxylysine, or glycosylated hydroxylysine (Bailey et al., 1974). Generally these Schiff bases are unstable, but more stable linkages develop (on shifting the double bond) in the formation of keto derivatives. Further dehydration and oxidative reactions, and the formation of complexes with imidazole of histidine results in very stable structures. It has been shown also that cross-linkages originating from hydroxylysine are relatively more stable than those formed by lysine (Bailey et al., 1977; Miller and Robertson, 1973). However, much information on the chemical nature of the cross-linkages has been derived from studies on tissue other than muscle. Little is known about the chemistry of cross- linkages of intramuscular collagen.

In summary, the increase in the mechanical stability and the progressive decrease in solubility of collagenous tissues in certain solvents coincide with gradual increase in intermolecular cross-linkages. The mechanisms of these changes is very complex, involving many variables like noncovalent interactions, dehydration, extent of glycosylation, packing of molecules, and location,

260 A . ASGHAR AND R. L. HENRICKSON

number, and chemical structure of cross-linkages (Tanzer, 1976). The hereditary disorders, such as the Ehler-Danlos syndrome and hydroxylysine deficiency, are reported to cause abnormal cross-linking in the dermis of affected individuals (Eyre and Glimcher, 1972; Mechanic, 1972). Apart from this, homocysteinuria may disturb the process of cross-linking in collagen due to interaction of homo- cysteine with the aldehyde group of polypeptide chains (Kang and Trelstad, 1973).

F. INTERACTION WITH CARBOHYDRATES

It is well documented that certain carbohydrates are covalently bound with collagen as an integral part of its structure. Earlier studies reported the presence of different sugars, such as glucose, galactose, mannose, rhamnose, ribose, and fucose, associated with collagen. Some of these (glucose and galactose) were found linked with acid-soluble (Highberger et al., 1964) and insoluble collagen (Hormann, 1965), whereas other sugars were detected in the neutral salt-soluble collagen (Highberger et al., 1964). However, the prevailing view is that only glucose and galactose are covalently bound with collagen either as monomer or disaccharide in vertebrate tissue (Spiro, 1969). Other sugars may be impurities resulting from inadequate purification of the collagen samples. The presence of mannose and N-acetylglucosamine in the C-terminal region of pro-a1 chain has been reported (Guzman et al., 1978).

Divergent views also exist as to how the carbohydrate moiety is linked to collagen. On the basis of earlier evidence, Harding (1965) concluded that an ester linkage may form between the COOH group of aspartic acid residues and the OH function of the hexoses. Bensusan (1965) suggested that themgars may be linked covalently to the E-NH, group of lysine by forming N-D-glycosyl linkage or they may bond to the N-terminal amino acids. However, there is more evidence that the disaccharide of glucose and galactose (M. J . Spiro and Spiro, 1971; R. G. Spiro and Spiro, 1971a-c) is glycosidically linked to the OH group of hydroxylysine (and threonine and serine) in some invertebrate collagen (Lee and Lang, 1968). The presence of glycosyl hydroxylysine and galactosyl hydroxylysine at positions 87 and 681 in the a1 chain, and at positions 87 and 174 in the a 2 chain has been reported by many workers (Butler and Cunningham, 1966; Butler, 1968, 1970; Aguilar et al. , 1973). The linkage between hexose monomers with hydroxylysine residue in collagen is shown on the next page.

Different opinions have been expressed as to the functional role of carbohydrates in the collagen structure. According to some researchers, the bulky carbohydrate group might direct the regular stagger of tropocollagen molecules by requiring a particular “fit” (Morgan et al., 1970; Piez et al., 1970). Some workers say that carbohydrate could mediate cross-linking without being incorporated into a cross-link (Bailey et al., 1970; Spiro, 1972), whereas others have


H O I I1

NH,- CH,- CH- CH,- CH,- C- C

NH I I

‘2 Hydroxylysine residue

-0 H O ~ -Galactose Hw -Glucose

HO

H OH

assigned an interacting role to carbohydrate between collagen and mucopolysaccharides (Traub and Piez, 1971).

G . POLYSACCHARIDES OF CONNECTIVE TISSUE

The polysaccharides of connective tissue are not an integral part of the collagen molecule. They in fact constitute the aqueous phase (ground substance) of the extracellular space in which collagen fibrils are embedded. The ground substance is believed to perform many functions in the tissues (Schubert and Hamerman, 1968). It provides the actual homeostatic environment of the cells and takes up their metabolites. It acts as a barrier against bacterial invasion. Besides, marked changes occur in the extracellular protein during differentiation, regeneration of cells, and hormonal action.

The polysaccharide complexes of connective tissue can be divided into two distinct groups: first, the glycoproteins consisting of protein molecules to which monosaccharides or oligosaccharides are covalently bound; and second, proteoglycans consisting of polysaccharide-protein complexes, in which the polysaccharide moiety makes up a major part of the whole molecule. The important polysaccharides found in association with various connective tissues are shown in Table 111, and their structures are depicted in Fig. 9. The molecular weight of these polysaccharides varies from 15 X lo4 to 15 X lo6, and the number of monosaccharide units in a molecule may vary from 50 to 50,000. Detailed information on the chemistry of proteoglycans and mucopolysaccharides is available in many excellent reviews (Muir, 1964; Fitton-Jackson, 1964; Schiller, 1966; Schubert and Hamerman, 1968; Merkel, 1971; Montreuil, 1980). The

I H U L b 111

THE MUCOPOLYSACCHARIDES (GLYCOSAMINOGLYCANS) ASSOCIATED WITH CONNECTIVE TISSUE

Sulfates/ period*

Polysaccharide (81 ycosaminoglycan) Synonyms Hexosamine Hexuronate Hexose Ester Amide Repeating unit

Hyaluronic acid Chondroitin Chondroitin 4-sulfate

Chondroitin 6-sulfate

Dermatan 4-sulfate

Keratan sulfate

Heparin

Heparin sulfate

__ -

Chondroitin sulfate A

Chondroitin sulfate C

Chondroitin sulfate B-heparin Kerato sulfate

Heparitin sulfate Heparin monosulfate

Glucosamine Glucuronate - 0.0 - D-Ghcuronic acid + 2-acetamido-2-deoxy-~-glucose

Galactosamine Glucuronate - 1.0 - D-(;hJcuronic acid + 2-acetamido-2-deoxy-4-O-sulfo-~- galactose

Galactosamine Glucuronate - 1.0 - D-(;hCUrOniC acid + 2-acetamido-2-deoxy-6-0-sulfo-~- galactose

Galactosamine Iduronate - 1.0 - L-Iduronic acid + 2-acetamido-2-deoxy-4-0-sulfo-~- galactose

Glucosamine - Galactose 1.0 - D-(;ahCtOSe + 2-acetamido-2-deoxy-6-O-sulfo-~- galactose

Glucosamine Glucuronate - 1.5 I .O D-Giucuronic acid f 2-deoxy-2-sulfoamino-o-g~ucose (both residues can also contain 0-sulfate groups)

Glucosamine Glucuronate - 0.5 0.5 D-Giucuronic acid + 2-deoxy-2-sulfoamino-o-g~ucose (also containing 0-sulfate) and D-glucuronic acid + 2-acetamido-2-deoxy-~-glucose

D-GhCUrOniC acid + 2-acetamido-2-deoxy-~-galactose

from Schubert (1964).


polysaccharide components of proteoglycans containing amino sugars coupled to either a neutral sugar residue or a uronic acid are termed glycosaminoglycans. Other names used for defining the compounds are deoxyglycosylaminoglycans and glucuronyldeoxyaminoglycosyl glycans (Schubert and Hamerman, 1968).

As can be seen in Fig. 9, the basic unit of mucopolysaccharides (glycosaminoglycans) is closely related to glucose, linked by glucosidic bond. In fact, each polysaccharide is composed of two different saccharide units, which repeat alternatively along the chain without branching. In all cases one of the saccharide moieties is hexosamine (mostly D-glucosamine, in some cases D-

galactosamine), whereas the other unit is generally a uronic acid derivative (either D-glucuronic or L-iduronic acid). The NH, group of hexosamine is never free; it is either acetylated or sulfated. Consequently, these polysaccharides have a high negative charge density as carboxylate, ester sulfate, or amide sulfate (Schubert, 1964). At physiological pH they exist as polyanions and are generally associated with an equivalent amount of Na+ counterion. However, in the native tissues the counterions may be of several kinds depending on the salt content of the surrounding tissue. The accumulation of counterions in close proximity to the polyanionic chain reduces the net charge of the mucopolysaccharides. This effect is called “shielding.”

Hyaluronic acid is one of the major polysaccharides of the ground substance. It is an unsulfated glycosaminoglycan, composed of D-glucuronic acid and N- acetylglucosamine linked alternately by p-1,4 and 1,3 glucosidic linkages. The chondroitin, heparin, and their sulfated derivatives have identical repeating units, consisting of glucuronic acid linked by 1,3 glucosidic bond to galactosamine, whereas the pairs are joined by 1,4 glucosidic linkage. Keratin is composed of glucosamine and galactose residues, sulfated at the C-6 position. Dermatin is a co-polymer of iduronic acid and galactosamine. The relative proportion of these mucopolysaccharides and their degree of sulfation vary among species, tissues, and with age (Hall, 1976).

In vivo the connective tissue polysaccharides do not occur free, but are linked to protein. Earlier studies indicated that polysaccharides are covalently bound to a globular protein through the OH group of the serine residue by way of galactose (Dorfman, 1964; Roden, 1965) to constitute the mucopolysaccharides. The latest structure that emerges for a mucopolysaccharide is that of a “bottle brush” in which the bristles may be regarded as the carbohydrate chains (35-65 in number) linked by a neutral trisaccharide to a protein core (Laurent, 1974; Phelps, 1974; Schubert and Hamerman, 1968). The protein core, consisting of 2000-3000 amino acid residues, has a globular portion and a linear portion, with a total length of 340 nm (Phelps, 1974).

On the other hand, Anderson and Jackson (1972) and Heikkinen (1973) have suggested some possible linkages between tropocollagen polymers via pro-


A

B

NHCOCH, H OH H NHCOCH, n

D-Glu&onic N - Acetyl-D- acid glucosamine

n 2500

n

D-Glu&Ironic N-Acetyl-b-galactosamine acid 4- sulfate

n = 60

C

n

L-Iduronic N-Acetyl-D-galactosamine acid 4- sulfate

D

n

D-Glucuronic N-Acetyl-D-galactosamine acid 6 -sulfate


E

F

COO@Na@

H l o H

-0 I H i!THSOpNa@

D-Glucuronic D-Glucosamine N-sulfate. acid 6 sulfate

n = 10-15

NHCOCH, H OH H N n c o c n :

265

1 An

n

D-Galactose N-Acetyl-D-glucosamine 6 -sulfate

n = 10-20

FIG. 9. Structure of the repeating saccharide units in different mucopolysaccharides of connective tissue. (A) Sodium hyaluronate; linkages p-1,3 and p-1,4. (B) Sodium chondroitin sulfate A; linkages p-1,3 and p- 1,4. (C) Sodium chondroitin sulfate B (dermatan sulfate); linkages p-1,3 and p-1,4. (D) Sodium chondroitin sulfate C; linkages p-1,3 and p-1,4. (E) Sodium heparitin; linkages CX-D-~,~. (F) Sodium keratan sulfate; linkages p-1,3 and p-1,4.

teoglycans, whereas other groups of protein may be linked through glycoproteins. According to Mathews (1970) proteoglycans, by virtue of a high negative charge, react directly with collagen to form macromolecular complexes having different biophysical properties and functions. Obrink (1974) stated that sulfated polysaccharides and proteoglycans (except keratin sulfate and hyaluronic acid) bind electrostatically to collagen under physiological ionic conditions and the binding increases with increasing chain length and charge density. According to Blackwell and Gelman (1974) the strength of interactions depend, in addition, on the side chain length of basic amino acid residues of collagen, or the degree of sulfation of the polysaccharide chain, and on the position and orientation of the sulfate and carboxyl groups of the polysaccharide chain. The intensity of the interaction between collagen and polysaccharide is also influenced by the pH of the medium, being highest at 3.0 for heparin and chondroitin sulfates A and C (Cundall et al., 1979), since they contain ester-sulfate ( R U S O , -) groups, which are not protonated at pH > 1 (Schubert and Hamerman, 1968). Hence, these groups can interact at pH > 1 with positively charged groups on collagen molecules.


H. IMMUNOCHEMISTRY OF COLLAGEN

The antigens (or allergens) in food are identified mostly with protein fractions. The ability of the food to incite immunological reaction is associated sometimes with the nature of protein molecules, their stability against physical agents (heat, cold, oxidation), H + concentration, enzymic action, and their ability to pass through the wall of the gastrointestinal tract with little or no alteration in molecular configuration (Straws, 1964). The allergy is an immunological phenomenon, considered as antigen-antibody reaction. The mediator of this phenomenon is probably histamine or a histamine-like compound, and the chain of reactions can be interrupted by histamine antagonists (Perlman, 1964).

Generally, the protein antigens have two operational immunological properties. One is called antigenicity, which denotes the capacity to interact with secreted antibodies, and the other is immunogenicity , which indicates the capacity of a protein to stimulate antibody synthesis. According to the current concept on the cellular basis of the immune response, the interaction of antigen determinants with antigen receptors occurs on the surface of antibody-producing precursor cells, called B cells. Optimal antibody production, however, requires for most antigens the cooperation between B cells and thymus-derived T cells (Katz and Benacerraf, 1972; Gershon, 1974). Probably macrophages also participate in this cooperation (Feldmann, 1974).

So far as the antigenic properties of collagen are concerned, a number of studies have shown that the major antigenic sites reside in the C- and N-terminal regions (Schmitt et al., 1964; Timpl et al., 1970, 1972, 1973; Furthmayr and Timpl, 1971; Rauterberg et al., 1972a; Becker et al., 1972, 1975b). It has been further shown that unoxidized or oxidized E-NH, groups of lysine in the terminal regions bind with antibodies (Rauterberg et al., 1972a). More detailed information on the immunochemistry of collagen and procollagen is available in a number of comprehensive reviews (O’Dell, 1968; Kirrane and Glynn, 1968; Timpl, 1976; Furthmayr and Timpl, 1976).

1. FUNCTIONS OF COLLAGEN IN TISSUES

Generally speaking, the main functions of fibrous biopolymers are structural, providing physical strength, cementing the cells together, and at the same time serve a sieve-like function for passage of metabolites from cell to cell (Bet- tleheim, 1974). In this respect, collagen is an unusual biopolymer, having a high tensile strength and being virtually inextensible. Nature, as Bailey stated, has utilized these unique characteristics to perform various mechanical functions in a variety of ways throughout the animal body. For example, in the case of skin and muscle, fascia collagen acts as a flexible network to contain other tissues in register. In the case of tendons and ligaments, collagen not only connects one tissue to another but also transmits tensional stress and resists the compressional


force (Flint, 1973; Miller, 1980). It also imparts some degree of flexibility to calcified tissue like bone and a certain amount of structural rigidity to cartilage (Viidik, 1973; Gathercole and Keller, 1974; Phelps, 1974). It constitutes the fine membrane that functions as a filtration barrier in the kidney, and constitutes the channels for solid and liquid transport. In all these tissues, collagen has been found in different but highly ordered fashions to suit the registered mechanical function of the respective tissue. However, the optimum functioning of the various tissues depends not only on the amount and types of collagen but also on the relative proportion of other associated constituents (Bailey, 1974).

The mucopolysaccharides associated with collagens are largely responsible for the water sorptive and retentive capacity of the tissues. They also create osmotic pressures, at physiological concentration, of a magnitude that is important to the living organism (Bettleheim, 1974; Laurent, 1974). As the polysaccharides also bind counterions, they affect the diffusion of ions through the connective tissues (Preston et al., 1972). Some workers have suggested that mucopolysaccharides protect collagenous tissue from enzymic attack (Eyre and Muir, 1975b; Osebold and Pedrini, 1976). Etherington (1977) espouses the view that the type and quantity of associated mucopolysaccharides are important factors in determining the degree of resistance of the collagen molecules to enzymic vulnerability.

IV. METABOLISM OF COLLAGEN

The macromolecular components of collagen are synthesized in large part at a ribosomal-messenger RNA complex within the cytoplasm of a family of mes- enchymal cells, which include fibroblasts, chondroblasts, and osteoblasts (Fit- ton-Jackson, 1964). The fibroblasts are quite familiar cells which synthesize connective tissue as a part of the repair process or normal embryogenesis. How- ever, the existence of several genetically distinct (Y chains in the collagen from different tissues suggests selective gene expression for collagen biosynthesis in certain cell types (Grant and Jackson, 1976). Fibroblasts, osteoblasts, and odon- toblasts in cell culture synthesize mainly type I collagen. The same seems to be true for human smooth muscle cells (Layman and Titus, 1975), whereas human skin fibroblasts are reported to produce also a good proportion of type 111 collagen (Lichtenstein e f al., 1975). Chondroblasts produce only type I1 collagen, whereas the basement membrane is synthesized either by epithelial or endothelial cells.

A. BIOSYNTHESIS ON POLYRIBOSOMES

The latest information on the biosynthesis of collagen (procollagen (Y chains) at the subcellular level is available in a number of comprehensive reports (Martin et al., 1975; Brownell and Veis, 1975; Grant and Jackson, 1976; Prockop et al., 1976; Beachey et al., 1979; Harwood, 1979). There is now general agreement


that the a chains of all the procollagens are synthesized about 50% larger than the ultimate a chains of the collagen, and have peptide extensions at both the C- and N-terminal ends (Grant et al., 1972; Dehm et al., 1972; Byers et al., 1974; Tanzer et al., 1974; Fessler et al., 1975a; Harwood et al . , 1977; Williams et al., 1976). These extensions are designated as P,a and P,a chain, respectively (Martin et al., 1975). As depicted in Fig. 5, the P,a chain consists of three distinct structural domains: a globular NH,-terminal region, a central collagen- like region, and a short globular region (Becker et al . , 1977; J. H. Fessler and Fessler, 1978). The PCa chain has only globular conformation (Olsen et al., 1977). The cysteine residues in the N-terminal region create only intrachain disulfide bonds, whereas they create both inter- and intrachain disulfide bonds in the C-terminal region. The extraglobular peptide portions (Sherr et al . , 1973; Murphy et al . , 1975) containing sugars, cystine and cysteine amino acids (Fur- thmayr et al., 1972; Clark and Kefalides, 1976) on the terminal ends are enzymatically cleaved before the molecule is liberated into the extracellular space (Bornstein, 1974; Martin et al., 1975; J. H. Fessler and Fessler, 1978; Prockop et al., 1979).

The extra terminal peptides (sometime termed “registration peptides”) presumably facilitate alignment of the three cx chains, and permit the remaining molecule to coil into the in-register triple helix (Grant and Jackson, 1976). The presence of interchain disulfide bonds in the terminal regions also seems to play a role in helix formations (Schofield et al., 1974; Uitto and Prockop, 1974). It has been shown that the individual a chains of tropocollagen are assembled as single polypeptides rather than from short peptides (Bornstein, 1970; Vuust and Piez, 1972). The polycistronic mRNA synchronizes the proper balance of the synthesis of dissimilar tropocollagen subunits, that is, a 1 and a 2 chains, in the case of type I collagen (Vuust and Piez, 1972) at 2:l ratio (Kerwar et al . , 1972; Prichard et al., 1974; Harwood et al., 1975a). This suggests that the two mRNAs may be present in a similar ratio.

Further, the collagen precursors are synthesized by heavy polyribosomes with a sedimentation value 300 to 400 S (Lazarides and Lukens, 1971; Harwood et al., 1974a,d, 1975b; Pawlowski et al., 1975; Vuust, 1975) on tight membrane- bound polyribosomes (Rosbash and Penman, 1971; Harrison et al., 1974; Har- wood et al . , 1975b). It takes 6-7 min for the synthesis of pro-al(I), pro-a2, and pro-al(II1) chains in vivo (Miller et al., 1973), possibly due to the greater demand for glycyl-tRNA and propyl-tRNA in the translation of the long [-Gly-X- Y-1, triple sequence of the collagen molecule (Grant and Jackson, 1976). On the contrary, translation of mRNA globular proteins may need only about 2 min.

At present two hypotheses exist regarding the initial synthesis of a chain (s) precursor. According to one view, the precursor of the three chains may be synthesized initially as a very long single peptide chain (Church et al., 1971, 1974; Tanzer et al., 1974; Park et al., 1975), analogous to the synthesis of the


insulin polypeptide (Steiner et al., 1974), and then cleaved into three pro-a chains. There is, however, little evidence in favor of this concept. The other view, based on pulse-chase studies, supports simultaneous translation of pro-al(I) and pro-alt(1) chains (Vuust and Piez, 1970, 1972; Harwood et al., 1973, 1975a, 1977; Schofield et al., 1974; Uitto and Prockop, 1974; Byers et al., 1975; Fessler et al., 1975b) by monocistronic mRNA (Diaz de Le6n et al., 1977) or polycistronic mRNA (Harwood, 1979). According to Williamson (1969), generally there are two patterns of genetic control for heteropolymeric proteins. First, the cistrons coding for the different subunits may be adjacent on the genome, and hence give rise to a polycistronic mRNA which ensures a balanced production of the dissimilar polypeptide chains. Second, the subunits may be coded by separate cistrons, linked or unlinked, to give rise to monocistronic mRNA molecules, which are separately translated. However, the most precise control of balanced synthesis of dissimilar subunits is expected from polycistronic mRNA, which ensures balance and synchronization. But, the balanced production of subunits by different monocistronic mRNAs would be liable to disturbance.

Hydroxylation of proline and lysine and glycosylation of hydroxylysine are the important changes which occur after the transitional period at the subcellular level in pro-a chains (Cutroneo et al., 1974).

1. Hydroxylation of Proline and Lysine

As the mRNAs carry no codons for the hydroxy amino acids found in collagen, they must be synthesized during the posttransitional period by enzymic hydroxylation of proline and lysine residues, already incorporated in procollagen. Nonhydroxylated collagen molecules are also denoted as protocollagen (Grant and Prockop, 1972). Hydroxylation of proline (generally at the Y position) is brought about by prolyl hydroxylase (Cardinale and Udenfriend, 1974; Prockop et al., 1976) mainly as trans-4-hydroxyproline in different collagens (Fietzek and Kuhn, 1976), except in type IV collagen, where 3-isomers (at the X position) have been found in fair amount (Gryder et al., 1975; Adams and Frank, 1980).

Considerably divergent views exist as to the site of hydroxylation at the subcellular level (Bornstein, 1974; Prockop et al., 1976). The general consensus of opinion is that it occurs before the polypeptides are released from the ribosomes. Again, contrary to the initial finding that prolyl hydroxylase is a soluble cytoplasmic enzyme, it is now well documented that this enzyme is membrane bound, associated with cisternae of rough endoplasmic reticulum for hydroxylation (Harwood et al., 1974c, 1975b) before helix formation (Ryhanen and Kivirikko, 1974a,b).

The hydroxylation process also requires Fe2 + , ascorbate, and decarboxylation


of 2-ketoglutarate to succinic acid and CO, (Grant and Jackson, 1976) as follows:

[ Gly--X-Proln *- ascorbate [GI~-X-PJ;-],~

2-Keto- Suciinate glutarate

The same sorts of events are involved in the hydroxylation of lysine except that the reaction is catalyzed by lysyl hydroxylase (Guzman et al., 1975). Many research workers have shown that hydroxylation of the a chains is important for the formation of the triple-helical structure of collagen (Fessler and Fessler, 1978; Olsen et al., 1975; Harwood et al., 1974a,b, 1977).

Many studies have shown differences in the extent of hydroxylation of proline residues in rat and calf skins, with rat skin being more hydroxylated than the calf skin (Fietzek et al., 1972; Fietzek and Kuhn, 1973, 1974, 1975). The extent of hydration is positively related with the T , value of collagen (Jimenez et a/ . , 1973; Berg and Prockop, 1973).

2 . Glycosylation of Hydroxylysine

According to Grant et al. (1975), the disaccharide a-D-glUCOSyl (1,2)-0-p-D- galactose, associated with collagen, has a significant bearing on its functional and structural properties. This sugar is linked with hydroxylysine (at the Y position in the collagen chain) by the glucosidic bond (Morgan et al., 1970; Aguilar et al., 1973), as mentioned earlier in Section III,D,5.

Two enzymes, namely collagen UDP-galactosyl transferase and collagen UDP-glucosyl transferase, catalyze the glycosylation of pro-a chains (Spiro, 1972; Risteli and Kivirikko, 1974; Myllyla etal. , 1975) in the presence of Mn2+ as cofactor (Myllyla et al., 1975). These enzymes are bound to the internal surface of rough endoplasmic reticulum cistemae (Harwood et al., 1975b), where glycosylation of peptide chains takes place (Brownell and Veis, 1975). As glycosylation could not be achieved with triple-helical collagen in in vitro studies (Myllyla et al., 1975; Risteli et al., 1976), in vivo glycosylation probably occurs before the procollagen chains attain the triple-helical structure in the cistemae of the rough endoplasmic reticulum. Several pathways have been proposed for onward transportation of procollagen (Weinstock and Leblond, 1974; Prockop et al., 1976). However, various studies suggested that the procollagen molecules are then transferred to smooth endoplasmic reticulum (Harwood et al., 1977), and then directed into the Golgi apparatus (Nist et a/., 1975; Olsen et al., 1975), from where they are possibly released by exocytosis through microtubules. Fig- ure 10 presents the overall events of the biosynthetic process of procollagen a chains and their transport into the extracellular spaces at the subcellular level.

NH2 - - COOH

N-protease

f

I C-protease

I

Fibril formation Aldehyde formation Intermolecular cross1 inking

FIG. 10. Schematic representation of the sequential events in the biosynthesis of procollagen at the subcellular level and subsequent polymerization in the extracellular space following stepwise cleavage of terminal extensions at NH, and COOH ends. From Grant and Jackson (1976). Courtesy of The Biochemical Society, London.


The polymerization of procollagen monomers to form collagen fibrils takes place stepwise in the extracellular space. It appears that the NH,-terminal region is cleaved first (Morris et al., 1975; Davidson et al., 1975), followed by stepwise removal of the C-terminal extensions of the three a chains of procollagen, consequently giving rise to an insoluble collagen polymer (Byers et al., 1975; Fessler et al., 1975b; Grant and Jackson, 1976). The cleavage of N- and C- terminal extensions is brought about by either the same enzyme or two different enzymes, presumably bound on the outer cell surface (Davidson et al., 1975). Freshly assembled collagen fibrils have little tensile strength, which increases due to the formation of more covalent bonds and cross-linkages (Grant and Jackson, 1976) with maturity as a function of chronological age.

It is obvious from the preceding review that biosynthesis of collagen involves a large number of posttranslational reactions. They can be influenced not only by defects in transcription and translations, but also by defects in any of the enzymes involved in the posttranslational steps. Consequently, cells that use the same mRNA may produce collagen of different types. A number of genetic defects in collagen synthesis have been described by Prockop et al. (1979) and Lenaers et al. (1971).

B. CATABOLISM OF COLLAGEN

Earlier studies have indicated that depolymerization of native tropocollagen macromolecules occurs by cleavage of certain bonds in the nonhelical region (Hodge et al., 1960; Hodge and Schmidt, 1960). For example, pepsin or trypsin attacks the region rich in tyrosine and acidic amino acids, and devoid of hydroxyproline (Steven, 1963; Rubin et al., 1963). The prevailing view is that nonspecific peptide hydrolases such as pepsin and chymotrypsin are not capable of disrupting the helical conformation of collagen (Stark et al. , 1971b). Howev- er, chymotrypsin (Bornstein et al., 1966) and lysosomal isoenzyme (cathepsin B) can convert p and y aggregates of soluble collagen to a chain (Burleigh et al., 1974; Etherington, 1976, 1977) by hydrolyzing the peptide linkage between glycine-isoleucine, or between serine and valine of the a 1 chain in the NH,- terminal region. Figure 11 shows the various cleavage sites on the polypeptide chain of a protein by some important proteolytic enzymes.

Harris and Krane (1973, 1974) have shown that collagenases hydrolyze the native collagen molecule at a point about % of the length from the NH,-terminal end. Different explanations have been given to explain the mode of action of collagenases on a1 and a 2 chains (McCroskery et al . , 1973; Gross et al., 1974). According to Weiss (1976), the cleavage site is at the NH,-terminal end of the nonproline helical portion of the molecule between two regions of tripeptides of opposite symmetry of their proline and hydroxyproline content. Thus, initial breaks produce two fragments with lower T , and higher solubility than the intact molecule, without any loss of helical structure (Gross and Nagai, 1965; Sakai


-Arg-Glv- Leu-Hyp- -Ala-Gly-Val- Ala-

b

CHYMO- 2 TRYPSIN 2

0 a 3

x

'1 '1 -Hyp-Gly-Glu-Hyp- -Ser-Tyr-Gly-Tyr- -GLy-Tyr- AspGlu-

N-Terminal region of a-chain N-Terminal region of o-chain -Thr-Gly-lleu-Ser-VaI-Pro- -Gly-Ala-Hyp-Gly-Thr- Pro-Gly-Pro

-1leu-Gly-Gln

h 4

c

-Lys-Ser-Gly-Asp-

FIG. 1 1. for the action of certain important peptide hydrolases.

Some typical cleavage sites on native collagen and denatured collagen (gelatin) molecules

and Gross, 1967). On the other hand, Harris and Krane (1974) are of the view that collagenase cleaves through the collagen triple helix at one point without having definite specificity for denatured collagen products.

Bazin and Delaunay (1972) reported on a collagenolytic cathepsin, other than B I , which degraded insoluble collagen without affecting triple-helical conformation, whereas other workers have shown that cathepsin B, (EC 3.4.22.1) degrades insoluble collagen at pH 3.5 (StanEikovB and Trnavskjr, 1974). According to Etherington (1974), lysosomal cathepsin B I removes the telopeptide regions. However, increase in the extent of intermolecular cross-linkages renders collagen resistant to collagenase (Harris and Ferrell, 1972).


The studies of many workers (Harris and Krane, 1974; Woolley et al., 1975a,b) have provided strong evidence which indicates that soluble type I1 collagen is degraded by collagenase, but the rate is much slower than for type I collagen. The action is probably hindered by the presence of the covalently bound bulky carbohydrate moiety to the hydroxylysine residue in the a 1 (11) chains. There is also evidence that collagenases from different tissues cleave collagen at different sites at different rates (Jeffrey and Gross, 1970; Tokoro et al., 1972; Davison and Berman, 1973; Wahl et al., 1975).

A critical evaluation of various studies by Weiss (1976) suggests that denatured collagen is easily catabolized by such noncollagenous peptide hydrolases as trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.1), pepsin (EC 3.4.23.1), papain, gelatinases (EC 3.4.24.3), and clostridiopeptidase (EC 3.4.24.3). The native collagen is quite resistant. The unusual primary structure of native collagens, and the high levels of proline, hydroxyproline, and glycine residues make collagen an unsuitable substrate for most of the exopeptidases. However, a variety of noncollagenous endopeptidases can cleave certain regions of the molecule, high in either polar or nonpolar residues and low in imino acid content. This gives rise to larger fragments which possibly are not degraded further (Weiss, 1976). Hence, many of the earlier studies, quoted by Piez (1967) and Eisen (1969), where experimental conditions preclude the possibility of denaturation of collagen, are invalid. For instance, some studies have claimed excessive degradation of insoluble collagen by collagenase, which may be due either to rupture of intermolecular bonds by some pretreatment or to contamina- tion of the enzyme preparation with another protease (Weiss, 1976). This is quite apparent from the study by Fugii and Kobayashi (1970), showing that first dissociation of acid-labile intermolecular Schiff base cross-links in the telopeptide region occurs in an acidic media, which sets the peptides free and makes them available for enzymic catabolism. Swelling of collagen at acidic pH results from or is partly responsible for the cleavage of cross-links (Etherington, 1972). The conformation of collagen molecules also determines the rate of enzymic action (Hayashi et al., 1980).

More detailed information on in vivo degradation of collagen may be found in a number of reviews (Lapikre and Gross, 1963; Eisen, 1969; Harris et al., 1970; Davison, 1973; Perez-Tamayo, 1973, 1979; Harris and Krane, 1974; Gross, 1976; Weiss, 1976; Harper, 1980).

V. FACTORS AFFECTING COLLAGEN COMPOSITION AND STRUCTURE

Some in vitro studies have indicated that environmental factors can influence the mechanism for gene selection to produce a particular type of collagen (De- shmukh and Kline, 1976; Mayne et al., 1976; Deshmukh and Sawyer, 1977).


Many in vivo studies have shown changes in collagen composition and structure by ante- and postmortem factors. An account of those factors is presented in this section.

A. ANTEMORTEM FACTORS

A number of antemortem factors such as sex and sex condition, caloric and nitrogen intake, hormonal status of the animal, age, and dietary components have a significant bearing on the composition of collagen. The available information on these aspects is discussed below.

1. Sex and Sex Condition

The amount of collagen in tissues seems to be influenced by simultaneous activity of anabolic and catabolic systems between sexes. Prost et al. (1975) reported a relationship between the content of connective tissue and sex of the animal, indicating that bovine males have more intramuscular connective tissue than females. A similar trend was found in skin collagen from male and female rats (Hall et a l . , 1974). However, Summers et al. (1978) observed no real difference in the amount of muscular collagen of ewes and wethers.

2 . Caloric and Nitrogen Intake

Gross’s (1954, 1964a) study has shown that salt-soluble collagen disappeared on prevention of growth in rats by caloric restriction. The findings of McCance and his colleagues (Dickerson and McCance, 1960; Widdowson et al., 1960) indicated that extracellular protein content (collagen) increased in the avian and pig skeletal muscle as a result of undernutrition. Similarly Mendes and Waterlow (1 958) reported that muscle connective tissue continued to increase in amount during the depletion period of young rats by underfeeding. Other workers (Hark- ness et a l . , 1958; Summers and Fisher, 1960) have noted similar effects of inadequate diets on body collagen. According to the work of Hagan and Scow (1957) and Montgomery et al. (1964), short-term starvation caused a greater reduction in the sarcoplasmic proteins than in the myofibrillar proteins. The connective tissue remained relatively unaffected.

In a study of the influence of zero- and negative-energy balance feeding on different protein fractions of muscle from growing lambs, Asghar and N. T. M. Yeates (1979) found an increase in total stroma protein, including the alkali- soluble and alkali-insoluble stromal fraction. Besides the changes in amount, the physicochemical nature of the stroma fraction was also altered by nutritional stress, whereby the extent of acid-stable cross-linkages significantly increased. Another study by Asghar et al. (1981) on growing rabbits indicated similar effects of maintenance and submaintenance feeding on L. dorsi stromal protein


(collagen). A number of studies have also been reported on the connective tissue as it is influenced by breeds, feeding regimens, and management practices with hope of relating such information to meat tenderness (Wierbicki et a l . , 1955; Paul, 1962; Sharrah et a l . , 1965). The collagen content varies with type of muscle, being more in “slow” than in “fast” muscles (Kovanen et a l . , 1980).

3. Effect of Vitamins and Minerals

Among the water-soluble vitamins, the role of ascorbic acid in synthesis and maintenance of collagen has been studied intensively (Gould, 1968; Levene et a l . , 1974). There is general agreement that vitamin C deficiency causes a decrease in collagen synthesis without increased catabolism (Robertson, 1964). Gross (1954) believes that the basic defect occurs before the level of synthesis of the collagen molecule, and not in the formation of intramolecular bonds. It seems that hydroxylation of proline and glycosylation of hydroxylysine are impaired in the absence of vitamin C (Barnes, 1975; Berg et a l . , 1976), resulting in a higher hydroxylysine-to-hydroxyproline ratio in pro-a chain than normal. Staudinger et al. (1961) gave a possible mechanism to account for the role of vitamin C in free radical formation and in hydroxylation of collagen. This involves electron transport in microsomes. According to them, the first product of the reaction with oxygen is an OH radical which may enter into the hydroxylation reaction. In some cases hydroxylation may also involve vitamin C-dependent NAD-oxidase as follows:

Trans- Dehydro- Ascorbic acid ’?> hydrogenase (flajin) ( ascorbic acid )Cytochrome oxidase b, ZOH-H,b + %02

NAD+ Ascorbic acid I (.1* Other studies have indicated that the oxygen of the OH group of hydroxyproline is derived from molecular 0, and not from water (Fujimoto and Tamiya, 1962; Prockop et a l . , 1962a).

According to Gould (1968), certain B-complex vitamins are also believed to influence the collagen synthesis either by their involvement in NADP in the proline hydroxylation chain reaction or in Krebs cycle intermediates. He has quoted some studies indicating that deficiency of vitamins B, and B, decreased the synthesis of collagen in the skin of rats.

Among the fat-soluble vitamins, deficiencies of vitamins A and D have been noted to affect the synthesis of collagen. Vitamin A deficiency seems to depress collagen synthesis (Robertson and Gross, 1954). Chung and Houck (1964) reported that hypervitaminosis A decreased salt-soluble collagen, and increased the


acid-soluble fractions without any effect on the insoluble collagen. However, the hydroxylation of proline is not impaired by hypervitaminosis A (Dingle et al., 1966). A toxic dose of vitamin D increased the amount of both collagen and mucopolysaccharides, but Clark and Smith (1964) noted a decrease in mucopolysaccharides. Vitamin E deficiency increased the content of soluble collagen without affecting the total amount, suggesting prevention or alteration in cross-linkage (Gould, 1968). The effect of fat-soluble vitamins may be indirect by influencing the membrane stability and permeability.

The information regarding the effect of dietary minerals on collagen synthesis seems very scanty. Some early studies have reported that dietary copper deficiency causes mechanical weakness in the framework of collagen (Coulson and Carnes, 1962; Kimball et al . , 1964).

It has been established that lysyl oxidase is a Cu2 + -containing enzyme, which oxidatively deaminates the E-NH, group of lysine and hydroxylysine to yield aldehydes, which form cross-linkages in collagen (Section 111,E). This may explain the weakening of collagen structures due to Cu2+ deficiency in the animal body. The deficiencies of Fe2 + , Mn2 + , and Ca2 + may produce a similar effect on collagen. The reason is that hydroxylation of proline and lysine residues in collagen brought about by prolyl and lysyl hydroxylases, respectively, is dependent on the presence of Fe2+ (Section IV,A). The glycosylation of hydroxylysine residues by galactosyltransferase and glucosyltransferase depends on Mn2+. The activity of amino protease and carboxyprotease, which cleave the P,a and P,a region for converting procollagen to collagen, requires Ca2 + (Fugii and Tanzer, 1977; Uitto, 1977; Tuderman et al., 1978; Leung et al., 1979). Hence, the deficiency of these minerals may impede in vivo biosynthesis of collagen.

4. Effect of Hormones

There is ample experimental evidence that the anti-inflammatory compounds cortisol, glucocorticoids (Dougherty and Berliner, 1968; Vogel, 1974), and prednisolone (Hall et al., 1974) inhibit the synthesis of new collagen fibrils and promote the removal of already formed collagen. Other studies have shown that these corticosteroids decrease the level of posttranslational enzymes which are involved in collagen synthesis (Oikarinen, 1977; Newman and Cutroneo, 1978). However, it has been difficult to establish whether their effect is specific. The adrenocortical steroids also inhibit the formation of mucopolysaccharides, probably by affecting fibroblasts. Castor and Prince (1964) found that glucocorticoids suppressed the metabolic activity of fibroblasts, including collagen synthesis. Kowalewski (1966) noted a decrease in saline-extractable, insoluble, and total hydroxyproline, and an increase in acid-soluble hydroxyproline on administration of corticosteroid. These effects were minimized by administering anabolic

27 8 A. ASGHAR AND R . L. HENRICKSON

androgen-like methandrestendone, HCTH, and 17-ethyl- 10-nortestosterone (Schiller and Dorfman, 1955), which antagonizes the action of corticosteroids.

On application of 4- 14C-labeled cortisol, corticosterone, and other adrenal corticosteroids, the hormones tended to localize in or on fibroblasts (Schneebeli and Dougherty, 1963) and to inhibit pinocytosis (Berliner and Nabores, 1967). A number of studies have shown a decrease in the uptake of sulfate by various tissues (Clark and Umbreit, 1954; Schiller and Dorfman, 1955; Kowalewski, 1966) by administering these hormones, which inhibit the synthesis of hyaluronic acid in the skin. Manner and Gould (1965) are of the view that hydrocortisone interferes in part with collagen synthesis on the ribosomes.

Hypophysectomy has been shown to decrease the uptake of [35S]sulfate in cartilage (Denko and Bergenstal, 1955; Murphy et al., 1956). However, according to Schiller (1966), both hypophysectomized and hypothyroid (thyroidec- tomy) conditions cause a decrease in condroitin sulfate but an increase in hyaluronic acid. These effects can be reversed by administering growth hormone and thyroxin, respectively. Thyroxin seems to increase the rate of [35S]sulfate incorporation (Dziewiatkowski et al . , 1957), whereas the thyrotropin and thio- uracil impair the effect of growth hormones. However, Lorenzen’s (1962) study suggested that administration of thyroxin itself caused a decrease in the content of hydroxyproline and hexosamine; their amount increased when thyroxin was injected in rats along with adrenaline. Baker et af. (1959) proposed that thyroid- induced alterations in connective tissue metabolism may be due to a failure of the pituitary to produce thyroid-stimulating hormone (TSH). Parathyroid hormone, presumably parathormone, affects the components of bone matrix as well as the mineral content. It is believed that parathormone has an inhibitory effect on the resorption of the metaphysis, or it may stimulate the production of chondroitin sulfate in the epiphyseal plate (Bronner, 1961; Sheltar et al., 1961). On the contrary, other studies reported an increase in the excretion of hydroxyproline into urine due to parathormone administration (Avioli et al., 1966), possibly by stimulation of collagenase synthesis (Harris and Sjoerdsma, 1966).

It has been observed that under diabetic conditions the incorporation of [ 14C]acetate and [35S]sulfate into hyaluronic acid and chondroitin sulfate decreased in skin (Schiller and Dorfman, 1955). This suggested that insulin appar- ently acts as a stimulant for the synthesis of mucopolysaccharides. The facts that insulin markedly enhances the pinocytosis in fibroblasts (Schneebeli and Dough- erty, 1966) and that glucose enters the cells by a pinocytotic process, may well explain the biochemical mechanism by which insulin stimulates mucopolysaccharide synthesis.

According to Davidson and Small (1963), the ratio of keratosulfate to chondroitin sulfate C increased as a function of age. However, the growth hormones, estrogen and endrogen, alter this ratio, as is found at younger ages. Estrogens tend to increase the hyaluronic acid content of connective tissue (Muir,


1965). Some workers have shown that estrogen inhibits resorption of meta- physeal bone (Budy et al., 1952). Similarly, testosterone also increases the amount of hyaluronic acid (Doyle et al., 1964). The growth hormone ACTH is believed to promote collagen synthesis, whereas STH increases the number of fibroblasts and their synthesis of protein in vitro (Dougherty and Berliner, 1968). Harkness (1961) stated in his review that the growth hormones estrogen and deoxycorticosterone favor collagen deposition, whereas parathyroid hormone, cortisol, and related steroids discourage collagen synthesis. On reviewing the role of hormones in connective tissue metabolism, Sinex (1968) concluded that a decrease in insulin, estrogen, androgen, and thyrotropin is likely to favor the formation of dense connective tissue, deficient in mucopolysaccharides. Increase in glucocorticoids and thyroxin also produces similar effects in connective tissue. The mechanisms by which this occurs can only be the subject of speculation. The hormones may act as cofactor, or stimulate a dormant enzyme, to affect the intermolecular bonds. By using [14C]leucine, Gabourel and Fox (1965) observed that hydrocortisone affected the mRNA-tRNA system, but that tRNA or soluble enzymes were not influenced, and that the total amount of RNA decreased. Manner and Gould (1 965) also found a significant decrease in the amount of material in the polysomal fractions. On reviewing the actions of steroid hormones at cellular and molecular levels, Grant (1969) concluded that these hormones possibly intervene at more than one point in the process of transcription, and probably not in the process of translation of the genetic message. Figure 12 summarizes the possible sites of action of steroid hormones at the subcellular level.

Actinomycin D Nuclear

Amino acid rncmbmne DO01 ,&-

FIG. 12. Diagrammatic presentation of possible sites of action of some steroid hormones and inhibitors at the subcellular level. From Grant (1969). Courtesy of The Biochemical Society, London.


5. Lathyrism

The phenomenon of lathyrism (osteolathyrism or odoratism) seems to have originated from the work of Ponsetti (1954), who first observed the deformities on feeding sweet peas (Lathyrus odoratus) to rats. Later studies indicated that p- aminopropionitrile (NH,-CH,-CH,-CN) was the causative lathyritic factor in sweet peas; the P-aminopropionitrile interferes with collagen metabolism and hence results in deformities (Levene and Gross, 1959). Further studies have shown that aminoacetonitrile and semicarbazide also exhibit a lathyrogenic effect. This aspect has been reviewed extensively by Tanzer (1965), Levene (1973), and Barrow et al. (1974).

Lathyrogens affect the metabolism either by increasing the solubility of collagen (Smith and Shuster, 1962; Tanzer and Hunt, 1964), or by causing defective synthesis of tropocollagen a chains (Martin et al. , 1963), which are incapable of forming inter- and intramolecular cross-linkages (Stelder and Stegemann, 1962; Tanzer, 1965). Gallop (1964) espoused the view that lathyrogens may react with COOH groups of the mucopolysaccharide moiety, and hence prevent them from cross-linking. Later studies have indicated that the a chains of collagen from lathyritic animals are normal and potentially capable of cross-linking, but in the presence of a lathyrogen, the E-NH, groups of lysine residues, which are involved in the formation of interchain bonds, are not oxidatively deaminated. In other words, lathyrogens specifically inhibit lysyl oxidase by irreversibly binding with enzyme (Narayanan et al., 1972; Gallop and Paz, 1975). Consequently, cross-links do not form (Page and Benditt, 1967; Miller and Matukas, 1969, 1974; Trelstad et al., 1970; Levene et al., 1972). It has been anticipated that lathyrogens block the 0-glycosidic linkage between the reducing group of hexoses and the peptide chains, and suppress the synthesis of desmosine and isodesmosine from lysine.

However, Edvin (1971) was granted a patent in which he claimed to have achieved increased solubility of muscle collagen from old cattle by administering a lathyrogen and hence produced tender meat. If so, the above proposal on the mechanism of lathyrogen’s action do not explain this effect. Again, whether or not a lathyrogen causes any interference in the synthesis of mucopolysaccharide has been a controversial point. Ross’s (1968) review of the literature on this aspect indicated variable findings. Some studies reported inhibition of [35S]sulfate incorporation into tissue, whereas others have shown increased uptake or little effect by administering lathyrogens. Barrow et al. (1974), in their review, differentiated between two types of lathyrisms, namely, neurolathyrism and osteolathyrism. The former occurs in animals and man, while the latter is specific only for rats and turkeys.


6. Thiolism

Like lathyric compounds, p ,P-dimethylcysteine (penicillamine) was found to decrease the strength and increase solubility of skin collagen when administered to rats (Nimni and Bavetta, 1965; Nimni, 1968; Nimni et al., 1972; Deshmukh et al., 1971). Other thiol compounds, such as cysteamine, produced similar effects on administration to rats (Dasler and Milliser, 1958; Harris ef al., 1974; Siegel, 1977). Harkness and Harkness (1966) also reported a marked decrease (to 10%) in the tensile strength of skin collagen (at pH 7.0-7.5) by thiol compounds even at lo-, M concentration. All of these compounds possibly act as irreversible lysyl oxidase inhibitors (Siegel, 1979).

The thiol compounds have been divided into two groups with regard to their effect on collagen (Nimni and Harkness, 1968). The first group of compounds has an NH, group adjacent to the SH group (e.g., cysteine, cysteamine, penicillamine) and is more effective in decreasing the tensile strength, possibly by shifting the pH toward the more alkaline range (> 9.5). The second group of compounds has either no NH, group or a blocked NH, group (e.g., N-acetyl- penicillamine, glutathione). These thiol compounds, contrary to those of the first group, tend to increase the tensile strength of collagen above pH 8.0. Halogen produces similar effects (Harkness and Harkness, 1965; Nimni and Harkness, 1968).

7 . Chronological Age

Hall (1 976) has discussed in detail the macrostructural, microstructural, chemical, and biochemical changes that occur in connective tissue as a function of chronological and biological age. Age-associated changes in the physicochemical stability of collagen have also been treated at length by a number of authors (Jackson and Bentley, 1960; Gross, 1961; Verzar, 1964; Bakerman, 1964; Sin- ex, 1968; Gutmann, 1977; Selmanowitz et al., 1977; Wada et al., 1980). There is agreement that aggregation and cross-linkages in collagen continue to increase with age, and eventually reach a point at which they become incompatible with normal physiological functions. Thus old collagen is tougher and less hydrated and has thicker and denser fibers than newly synthesized collagen (Gross, 1961). According to Sopata et al. (1974), stable cross-linkages form with time and insolubility results in about 3 weeks after the formation of tropocollagen units. Many other studies have reported decrease in solubility and increase in cross- linkages in collagen with age (Hill, 1966; Herring et al., 1967; Bailey, 1968; Shimokomaki et al., 1972; McClain, 1971; Bailey and Shimokomaki, 1971; Dutson, 1974).

282 A . ASGHAR A N D R. L. HENRICKSON

The quantity of hydroxyproline released (labile collagen) from tendon into Ringer’s solution on heating to 65°C for 50 min was reported to decline with an increase in biological age (Verzar, 1964). Similarly, Go11 et al. (1963, 1964a,b) found 42% labile intramuscular collagen in a 50-day-old calf, and only 2% in a 10-year-old bovine, and attributed this difference to the number and strength of cross-linkages in collagen with age. These observations were substantiated by many other workers (Vognarovi et al., 1968; Wada et al., 1980). A significant correlation between labile collagen and toughness of meat within ovine (Smith et al., 1968) and bovine maturity groups (Field et al., 1970a) suggested that differences in cross-linkage exist even within animals of the same maturity. The differential thermal analysis study by Field et al. (1970b) on hydrothermal shrinkage of collagen from L. dorsi and bicep femoris from the same animals indicated differences in collagen structure between muscles. These observations possibly can now be explained in terms of recently discovered different types of collagens (Section II,A,2).

Divergent views have also been expressed concerning the changes in collagen content (in muscle) as a function of age. Some researchers recorded an increase in collagen content with increasing age (Kim et al., 1967; Hunsley et al., 1971; Nakamura et al., 1975), whereas others observed little relation between age and the amount of connective tissue in muscle (Kauffman et al., 1964; Reagan et al., 1976) or in dermis (Hall et al., 1974). Still other studies showed decreasing trends in the amount of collagen in muscle with age, or the amount tended to become constant after the animal attained maturity (Wilson, et al. , 1954; Go11 et al., 1963; Hill, 1966; Kurosu, 1979).

Gallop’s (1964) proposition was once regarded as a possible explanation for the origin of the cross-linkages during aging. It has been stated herein (Section 111,D,2) that a subunits were believed to be linked through a pair of P-aspartyl ester bonds. During aging, the formation of P and higher aggregates from a chains, according to Gallop’s hypothesis, involved the rearrangement of the paired P-aspartyl ester bonds to link adjacent a chains so as to form inter- as well as intramolecular linkages. Although the review of literature presented by Hard- ing (1965) strengthened this view, it has not been proven experimentally (Sinex, 1968). Some workers (Deasy, 1962; LaBella and Paul, 1965) have suggested the participation of condensation products of tyrosine-oxidized residues in the formation of new cross-linkages. However, Sinex (1968) has questioned the experimental approach used to derive such conclusions.

More recent studies have shown that differences in cross-linkages do exist in insoluble collagen derived from various tissues (Bailey, 1968, 1970; Kang, 1972; Kang et al., 1970; Barnes et al., 1974; Robins and Bailey, 1973a; Bailey et al., 1969, 1970, 1973). Some of these cross-linkages (for example, dehydro- dihydroxy-lysinonorleucine) are acetic acid-labile, whereas others (such as hy-


droxylysine-5-ketonorleucine and glucosylamine) are acid-stable. The prevailing view is that the number of acid-labile cross-linkages decreases, whereas the number of acid-stable bonds increases with age in collagen from all tissues (Davison et al., 1972; Bailey et al., 1974). It is also of interest to note that the extent of hydroxylation of lysine, in newly synthesized type I collagen, decreases and that of proline remains constant with age of the animal (Barnes et al., 1974, 1976). The lysine residues in the N-terminal regions of al(1) and a 2 chains, which have a special role in forming intermolecular cross-linkages and hence stabilizing extracellular fibrils (Tanzer, 1973), are hydroxylated to the extent of 50% in embryonic tissues. However, a failure of hydroxylation at these sites occurs in skin collagen (Barnes et al., 1974). Hydroxylation of lysine in other regions of collagen molecules is not affected. Perhaps the changes in the relative distribution of types I and 111 collagen in dermis with age are significant. Both these types are present in an about equal ratio in fetal dermis, but the proportion of type 111 falls to 15-20% of the total collagen with advance in age (Epstein, 1974). If so, then it may involve turning off the particular gene which is responsible for the development of the mRNA for synthesizing type 111 collagen.

The findings seem to be contradictory as to the nature of changes with age in the carbohydrate content associated with collagen. Hormann (1965) reported a decrease in carbohydrate concentration, whereas Joseph and Bose (1962) reported an increase with age. The presence of hexosyl-lysine and hexosyl-hydroxylysine in aged tissue has been taken as indicative of linking of glycoprotein to collagen. This suggests another type of intermolecular cross-link which is age dependent and binds the collagen fibrils with glycoprotein molecules that en- velop these fibrils (Balazs, 1977). Among the changes in mucopolysaccharides with maturity of collagen, hyaluronic acid content decreases accompanied by an increase in sulfated glycosaminoglycans, especially chondroitin sulfate B (Loewi and Meyer, 1958; Muir, 1964) with a higher charge density (Mathews, 1964), and to some extent heparitin sulfate and keratosulfates (Kaplan and Meyer, 1960; Meyer et al., 1965). It is thought that the substitution of more viscous hyaluronic acid with less viscous sulfated mucopolysaccharides may favor thermal motion of collagen for interaction. It is also likely that chondroitin sulfate, which is very highly charged (Balazs, 1970a,b), may be more shielded by its own associated protein moiety, preventing interaction with collagen.

Age-associated changes in reducible components of bovine collagen have been reported by Robins and Bailey (1973a), whereas Mathews (1973) discussed the changes in glycosaminoglycan, indicating that the nonsulfated fraction increases, the sulfated chondrotin fraction decreases, and the ratio of heparitin sulfate to chondroitin sulfate increases with age. According to Heikkinen (1973), all of those factors which determine the particular metabolic activities of the fibroblasts such as oxygen consumption, the enzymic activity of the citric acid cycle,


glycolysis, and pentose phosphate shunt, together with those involved in synthesis of collagen, are depressed in old age.

B . POSTMORTEM FACTORS

1. Aging (Ripening)

A review by Asghar and Yeates (1978) shows that many attempts have been made to identify the nature of the postmortem changes in connective tissue (collagen). Those who followed changes in alkali-insoluble content of meat from different animals during aging, failed to identify any change (Wierbicki et al., 1954; Khan and Van den Berge, 1964; Herring et al . , 1967; Davey and Gilbert, 1968). Sharp’s (1963) study on collagen based on hydroxyproline estimations did not find any change in beef muscle connective tissue aged for 172 hr at 37°C under aseptic conditions. McClain et al. (1970) noted a decrease in the solubility of collagen from aged bovine muscle.

Other workers (Goll, 1965; Asghar, 1969) indicated that subtle conformational changes in collagen molecules on swelling under the influence of postmortem lactic acid accumulation may not necessarily affect the solubility characteristics of collagen. Sensitive procedures such as thermal shrinkage, electrophoresis, and susceptibility to collagenase digestion may well detect postmortem changes in collagen. By following such approaches many studies have demonstrated alteration in the collagen structure during aging of meat (Field et al., 1970c; Kruggel and Field, 1971; Pfeiffer et al . , 1972). Although Asghar and Yeates (1978) indicated that “acid-labile’’ bonds might be cleaved by the action of lactic acid on collagen during the ripening process of meat,‘the exact chemical nature of those cross-links still remains to be elucidated. Some studies also reported an increase in a components of collagen during aging due to cleavage of cross-links (Kruggle and Field, 1971; Wu, 1978). The study by Wu (1978) indicated that collagen type I is affected more during aging than type I11 collagen.

Divergent results have also been reported on whether or not the mechanical properties of collagen change with changes in pH. Although some workers found little effect on mechanical strength in the pH ranges 4-11 (Hall, 1951) or 5-8 (Partington and Wood, 1963), others reported a considerable drop in the strength of collagen in the pH ranges 7-4 and 10-12 (Harkness and Harkness, 1965). Harkness (1968) has further stated that at pH 6.0, the collagen strength was only 40% of the value at pH 7.5 and only about 20% at pH 5.0. Winegarden (1950) also found that strips of collagenic tissue, aged for 35 days, exhibited a slightly but consistently smaller shear force value than strips aged for only 10 days.


2 . Action of Lysosomal Enzymes

The earlier discussion (Section IV,B) has indicated that native collagen is resistant to proteolytic enzyme changes. However, denatured collagen can be hydrolyzed to low-molecular-weight components by a number of nonspecific proteolytic enzymes. In this regard lysosomal enzymes are believed to hydrolyze collagen which has been denatured first by lactic acid during aging. Go11 (1965) has reported the presence of collagenase in lysosomes which play a part in this process. This enzyme has the ability to attack the helical region of native molecules at physiological pH (Gross, 1970). Later studies have shown that cathepsin B, (a thiol-dependent lysosomal protease) can also degrade nonhelical regions of native collagen in the acidic pH range (Morrison et al., 1973; Burleigh et al., 1974; Etherington, 1976, 1977), whereas cathepsin G (Barrett, 1974) and cathepsin D (Parrish and Bailey, 1967; Dingle et a l . , 1971) accelerate the degradation of collagen and proteoglycan. Other lysosornal enzymes which affect mucopolysaccharides may also play a part (Canonico and Bird, 1970; Ono, 1970; Dutson and Lawrie, 1974).

3. Effect of Heating

Heating causes some protein denaturation whereby the noncovalent bonds stabilizing the quaternary, tertiary, and secondary structures of a protein are broken, and the highly organized macromolecules are distorted by the intense thermal collisions, the so-called Brownian motion. The word “denaturation” has been used in different contexts in the past to denote the changes in protein chemistry without any precise definition (Joly, 1955; Colvin, 1964). Presently, its use has been restricted to indicate only the alterations in the secondary or tertiary structure of polymers caused by any process (Kauzmann, 1956). Accord- ing to Jirgensons and Straumanis (1962), the coiled peptides become unfolded and the secondary bonds are loosened in the process of protein denaturation. These changes alter the properties of protein such as viscosity, optical rotation, X-ray pattern, chemical reactivity, and biological activity, including the exposure of SH groups and changes in shape of the molecule. Changes on the surface of the protein molecules, such as deamination or salt formation, are not involved in denaturation but electrostatic bonds are effected. The original conformation of the molecule remains intact. The coagulation or peptization may take place as secondary processes following denaturation (Ballou et al., 1944).

When heated in an aqueous medium, the collagen fibrils shrink. During this phase a part of the solvated water is lost (Fessler, 1965), possibly due to an increase of the hydrophobic interactions. Since the heat of adsorption of water decreases rapidly with increase in temperature (0-60”C), the adsorption of water


should decrease with increasing temperature (Wollenberg, 1952). As the temperature rises above the melting temperature (T,) of the crystalline regions, cohesive forces maintaining the orderly structure are weakened and the superhelix of collagen molecules collapses. The fibrous state of protein is labile from a thermodynamic viewpoint, and the globular form with random order (nonhelical) of chains is the stable configuration of a protein (Mirsky and Pauling, 1936). Thus, the inherent contractile tendency of the fibrils due to increased entropy results in a less orderly arrangement. Consequently, the transformation of collagen molecules into mixed random coiled components starts with continuing hydrothermal heating. The resulting product is a gelatin (von Hippel, 1967). According to Engel (1962) the denaturation of collagen proceeds in two stages. The helical structure is destroyed rapidly on heating, but separation of the chains takes more time. The chains can also be separated in part by warming the tropocollagen solution at pH 4.0 (Orekhovitch, 1958).

During the conversion of collagen to gelatin the amount of hydroxyproline in gelatin is inversely related to the maturity (age) of the collagen (Verzar, 1963, 1964; Gross, 1964b). The composition of gelatin is also influenced by some pretreatment of collagen (Gustavson, 1956). For instance, amide groups are little affected in acid-processed collagen, but alkali treatment destroys a significant amount of amide groups and salt linkages. The gelatin formed comprises fragments that have widely different molecular weights ranging from as high as 150,000 to as low as 10,000 (Pouradier and Venet, 1950). Veis and Drake (1963) had observed that gelatin is composed of such p aggregates as pI3, pI2, and p32. On the other hand, Worrall (1965) reported that salt- and acid-soluble collagen on heating at 37-60°C for 15 min gave rise to a and p components consisting of al, 1x2, p, and p2 types.

The thermal stability of collagen is considered to be directly proportional to the sum of proline and hydroxyproline (Gustavson, 1956; Harrington, 1964; Josse and Harrington, 1964; von Hippel, 1967; Piez, 1968). Rigby (1967) has indicated some inverse correlation between the thermal stability and serine content of collagen. Bailey and Lister (1968) attempted to identify the thermally labile cross-links in collagen.

The difference in the hydrothermal stability of sheep skin and bovine collagen is evident from shrinkage temperatures of 60 and 65"C, respectively. They require extensive acid or alkaline pretreatment and application of heat for gelati- nization (Gustavson, 1956). The zone of maximum hydrothermal stability is in the pH range 5-7. At high concentrations of H + or OH-, the T, of collagen is lowered. Low concentrations of neutral monovalent salts (0.1 M ) decrease the enthalpy of activation for denaturation of collagen molecules, and weaken the electrostatic bonds which are labile in aqueous salt solutions. However, divalent cations increase collagen stability (Adzet et al., 1979).


4 . Effect of Cooking Methods

A detailed account of the changes that occur in intramuscular connective tissue (collagen) during the cooking of meat has already been presented in an earlier review by Asghar and Pearson (1980). Various experimental evidence suggests that interstitial collagen partly dissolves during cooking. The extent of dissolution depends on the methods of cooking (roasting, broiling, deep-fat frying, pressure or microwave cooking), the duration of cooking, the internal temperature reached, and the maturity of the animal (age), which determines the extent of cross-linking in the collagen. Bayne et al. (1971) observed that only the alkali- insoluble collagen decreased during cooking of meat, whereas the salt-soluble fraction remained unaffected. Several scanning electron microscopic studies have revealed progressive denaturation (coagulation) of collagen fibers with increased internal temperature from 50 to 90°C during cooking of meat (Cheng and Parrish, 1976; Jones et ul., 1977; Leander et ul., 1980).

Extensive literature is available on the influence of heat processing on food protein quality in general (Altschul, 1958); however, little information has been reported with reference to collagen. Mauron (1972), on reviewing the effects of industrial and domestic processing on food protein quality, concluded that the nutritive value of protein is often improved by moderate heating. Intensive heating causes impairment and reduces the enzymic release of amino acids, especially in foods low in carbohydrates (e.g., meat). The presence of reducing sugars and other aldehydes and autoxidizing fat greatly contributes to heat deteri- oration of protein, whereas high water content reduces the incidence of heat damage.

VI. FUNCTIONAL PROPERTIES OF COLLAGEN IN FOOD SYSTEMS

Proteins have no parallel in their structural and textural versatilities. Although nature has designed proteins to perform specific roles in situ, they can display multifunctional properties by appropriate manipulations and processing treatments in different food systems. The functional properties depend on such intrinsic physicochemical characteristics of proteins as amino acid composition and sequence, molecular weight, conformation, and charge distribution on the molecules. The nature and charge density facilitate interactions with other food components such as water, ions, lipids, carbohydrates, vitamins, color, and flavor constituents depending upon the environment (pH, ionic strength, temperature) during preparation, processing, and storage. The functional properties are important for the organoleptic quality of the ultimate product. Different workers (Her-


mansson, 1975; Kinsella, 1976, 1979; Wolf, 1970; Chou and Morr, 1979; Shen and Morr, 1979) have treated these aspects in detail.

Though the physicochemical bases of some functional characteristics of proteins are little understood, proteins are not generally functional in the absence of an aqueous phase. Hence, hydration is the first and most critical step in imparting other desirable functional properties (Circle and Smith, 1972), such as swelling, gelatin, solubility, viscosity, wettability, emulsification, cohesion, adhesion, elasticity, and foaming in a food system. These properties of a protein are directly related to the manner in which the protein interacts with water in the product. Thus, the nature of the protein-water interactions in general, and collagen-water interactions in particular, are considered.

A. WATER BINDING

It is appropriate to consider first the fundamental principles governing the interaction of water molecules with other compounds and protein hydration in general. Water molecules have a unique three-dimensional geometrical structure due to hybridization of two lone pairs of valence electrons in 2s and 2p atomic orbitals of oxygen. The two electron pairs forming the covalent bond are attracted by the nuclei of oxygen and hydrogen, and the other two lone pairs of electrons are attracted only by the oxygen nucleus (Franks, 1975a,b). Thus, the water molecules possess dipolar characteristics due to asymmetric distribution of electrons. The dipolar nature of water molecules facilitates interaction not only with electrolytes carrying positive and/or negative charges, but also with strong electronegative atoms such as nitrogen and oxygen present in different functional groups in the components of a food system. The dipolar character and orientation of water molecules are also responsible for the very high dielectric constant, which lowers the electrostatic attraction between charged ions by forming stable hydrated shells around them. Natural water is composed of H,160 molecules, with small amounts of H,l80, H,I7O, and HDO. According 'to Pauling (1940), four resonance structures of water molecules are possible, of which the following three dominate:

H H+ H- .. :O: H' .. :0: H- .. : 0:

H ..

Biophysically, the protein holds water in two forms. One is called the bound, structural, or protective form and the other the free or biologically active form (Hamm, 1975; Fennema, 1976). The bound fraction (0.15-0.28 g/g protein) is firmly held as water of hydration by functional groups of the protein in the form of mono- and multimolecular layers, having ice-like structure (Wismer-Ped-


ersen, 1971). There is wide disparity among different workers regarding the amount of bound water associated with various functional groups. For example, according to Pauling (19454, each polar group on a protein molecule binds one H,O molecule with the exception of those COOH groups which are hydrogen- bonded to an amide group of glutamine or asparagine. Bull and Breese (1968) reported the binding of six H,O molecules per polar group. Speakman (1944) found the extent of water binding to be in the descending order of polar side chains, amino groups, carboxyl groups, and hydroxyl groups, followed by peptide linkages at intermediate activity of water and finally by the formation of multilayers at higher activity of water.

Kanagy (1950) stated that hydration of a protein occurs in steps of various energy content, as hydration involves functional groups of different degrees of strength and reactivity. The deuteron magnetic relaxation study on biopolymers by Glasel (1970) showed that uncharged carboxyl and amide groups interact strongly and imide groups weakly, whereas hydrophobic groups interact little with water. But, Karmas and DiMarco (1970) proposed the involvement of nonpolar amino acid residues in the binding of water. On the other hand, Bull and Breese (1968) associated water binding with the sum of the polar amino acid residues minus the amide groups as expressed by the following equation:

Y = -0.97 x lop3 + 6.77X, - 7.63X2,

where Y is moles of bound water per gram of protein at 25°C (RH, 0.92), X is the sum of moles of acidic, basic, and hydroxyl groups per gram of protein, and X, is the sum of moles of amide group per gram of protein.

A more comprehensive assessment of the water-binding capacity of different amino acids has been made by Kuntz (1971, 1975), based on nuclear magnetic resonance (NMR) studies. Table IV presents the data along with the pK values, isoelectric points (pl), and structure of different amino acids. It shows that the water-binding capacity varies with the nature of the amino acids and the charge at different pH values. Generally, cationic (lysine, arginine, histidine) and anionic (aspartic and glutamic acid) amino acids bind the highest amount of water followed by neutral ones, whereas hydrophobic amino acids bind little water (Kuntz and Kausman, 1974). Kuntz (1975) derived the following equation to estimate the extent of bound water on the basis of the nature of side residues of amino acid in a protein:

where A is grams of bound water per gram of protein;f,,f,, andf, are fractions of charged, polar, and nonpolar amino acid residues, respectively.

So far as the free water fraction is concerned, it exists in an ordered form


TABLE IV HYDRATION CAPACITY OF AMINO ACIDS AS DETERMINED BY MAGNETIC RESONANCE STUDIE

OF POLYPEPTIDES AND pK VALUE OF AMINO ACIDS<’

Hydratio PK (moles

H20/mo Amino acid Symbol Structure a-COOH a-NH2 Side group PI amino aci

A. Negatively charged form

Aspartic acid

Glutamic acid

Tyrosine

B. Positively

Arginine charged form

Histidine

Lysine

C. Neutral or uncharged form

Aspartic acid

Glutamic acid

Tyrosine

Asp ~

Glu ~

Tyr -

Arg +

His +

Lys +

ASP

Glu

TYr

Ionic

\ 0

p H , 0

0 \

0 //C-cH2-cHA

H I C-COO- 1.88

I NH,

H I .c--coo- 2.19 I F3

I H I I

I1 ( 1 I NH,

I H ’ I

HC=C-CH,+C-COO- 1.78 I I f I

H ‘ H + I I

1 1

H,N-C-NH-(CH,),+C-COO- 2.18

F2 I +

H+NH jNH,

H,N-CH, -(cH,),+c-coo- 2.20

; N H 3

HO ! H

4 I I 0 j NH,

HO j H

‘C-CH,+A-COO- 1.88

\C-CH,-CH,tC-COO- I I 2.19 $ 1 I N H 3

4 0

9.60

9.67

9.11

9.09

8.97

8.90

9.60

9.67

9.11

3.65

4.25

10.07

13.20

5.91

10.28

3.65

4.25

10.07

2.8

3.2

5.7

10.9

1.6

9.1

2.8

3.2

5.7

6.0

7.0

1.5

3.0

4.0

4.5

2.0

2.0

3.0


TABLE IV (Continued)

Hydration

HzO/mole PK (moles

iino acid Symbol Structure a-COOH (r-NH2 Side group pl amino acid)

4rginine

,ysine

Zysteine

Asparagine

3lutamine

Serine

Threonine

H ydrox yproline

I H : I

I1 D l NH I NH,

I + / H # I LYS H,N-cH,-(cH,),+c-coo- 2.20 I ' I NH,

Arg H,N-C-NH-(CH,),tC-CO- 2.18

Polar or hydrophilic

i H $ 1

HS- CH, + c - COO- 1.71 CYS I l j y 3

H P I H I 1

# I I NH, It

Asn 'C- CH,? C --COO' 2'02 4 0

HZN, ! H I I

-C-CH~-CH,fC-COO- 2.17 ' I : +

I H ' I

I

: NH, 4 Gln 0

Ser HO--CH,+C--COO- 2.21 j yH3

OH; H I I I

I I I Thr CH,-C+C--COO- 2.16

H ;y3 H

I HO-C4-,CHZ 1.82 I

Pro-OH H, +H--coo-

I H

9.09

8.90

0.78

8.80

9.13

9.15

9.12

9.65

13.20

10.28

8.33

-

-

-

-

-

10.9 3.0

9.7 4.5

5.0 1.0

5.4 2.0

5.7 2.0

5.7 2.0

5.6 2.0

5.8 4.0

(continued)

292 A. ASCHAR AND R. L. HENRICKSON


Amino acid

Phenylalanine

Clycine

Valine

Leucine

Isolucine

Methionine

Alanine

Tryptophan

Hydration

HzO/mole Symbol Structure a-COOH a-NH:, Side group pl amino acid

PK (moles

Nonpolar or hydrophobic

I H I I

HfC-COO- 2.34

I +

H3C\ i 7

H3C, ! H

Val CH’C-COO- 2.32 / ; I H3C M I 3

I 1

CH-CH I C-COO- 2.36 zT I Leu /

H3C : NH3

Ile CH,-CH,-CH+C -COO- 2.26

I H I I

I I 1

CH3I MI,

I H I i ‘ I

Met CH,-S- CH2-CH2+C- COO- 2.28

j I y3

I H ! I

i NH, Ala CH3+ C -COO- 2.34

I

I + ! H

Trp @f;cH2-g3coo- : I 2.38

I

H

9.13 - 5.5 0.0

9.60 - 6.0 1.0

9.62 - 6.0 1.0

9.60 - 6.0 1.0

9.62 - 5.9 1.0

9.21 - 5.7 1.0

9.69 - 6.0 1.5

9.39 - 5.9 2.0

(continue



Hydration

HzO/rnole PK (moles

nino acid Symbol S ttucture a-COOH a-NH2 Side group pl amino acid)

OHydration values are taken from Kuntz (1971). Values were determined in the pH range 6 8 at -35”C, except that ilues for uncharged Asp and Glu were measured or extrapolated at pH 4.0, for uncharged Arg at pH 10.0, for charged Lys at pH I@-11, and for charged Tyr at 12.0. The pK and p1 values are taken from Nivard and Tesser 965).

(because of H,O-H,O molecule interaction) with either restricted motional freedom (Ling and Walton, 1976; Ling, 1972) or freely mobile (Cooke and Wien, 1973; Cooke and Kuntz, 1974). This fraction is greatly affected by the spatial structure of the protein, changes in electrostatic forces of the ionic groups, and hydrogen bonding (Hamm, 1975). Changes in the pH value and ionic strength of the medium affect the conformational state of a protein and hence influence the binding sites, making them sterically available or unavailable for interaction with water. The transition of a protein molecule from compact globular to a random coil conformation exposes buried side groups for water binding. However, those proteins which are closely packed, strongly H-bonded, and devoid of ionic groups (e.g., silk fibroin) or stabilized by disulfide linkage (e.g., keratin) are resistant to hydration (Leeder and Watt, 1965). Some of the important biophysical factors governing the binding of “free” water by proteins will now be presented.

I. Effect of H+ and OH- Ions

A variety of chemical bonds and other forces determine the spatial configuration and conformation of proteins (Jones, 1964). Table V provides a summary of the structural forces, interacting groups constituting various chemical bonds, their energies, and interacting distance. The changes in protein conformation, that is, unfolding of helical structure, can be considered in terms of free energy of these forces by the following equation (Scheraga, 1963):

TABLE V STRUCTURAL FORCES IN PROTEINSU

Distances of Energy interaction Interacting

Mechanism (kcalimole) (A) groups Example

Covalent bond Electron sharing 3& 100 1-2 C-C, C-N, C=O, C-H C-N-C

s--s P

C<\ +

- -NH,+

-c=m I I

Ionic bond Coulomb attraction between 1&20 2-3 charged groups of opposite sign N H z

-coo- HN-. f--NH

H '.$!--

2-3 N-H.. . o=c on.. . NH;..; NH;..; NH...; coo-. . .

Apolar groups

Hydrogen bond Hydrogen shared between two 2-10 electronegative atoms

Van der Waals attractive Mutual induction of dipole mo- 1-3

groups Electrostatic repulsive Coulomb repulsion between 4142/r2

charged groups of like sign

force ments in electrically apolar

force

forces groups in close proximity Van der Waals repulsive Repulsion between apolar llrl2

3-5

Intraresidue bonds Peptide Disulfide a-Amino group Lysine Arginine Histidine Aspartic glutamic a-Carboxyl group Amide-carbonyl group Serine, threonine, tyrosine Polar side chains of residues

Apolar side chains

Polar groups of like sign

Steric hindrance between All groups

Polar side chains

side chain groups

oFrom Jones (1964), courtesy of the AVI Publishing Company, Inc., Westport, Connecticut.


where WUnf is the standard free energy of unfolding of the protein structure, and Fob, F", pH, FRO, Ecomb, and FElect represent the free energy of unfolding of the helical backbone, of covalent cross-linkages, of H bonding, of hydrophobic bonding, of combination of randomly coiled chain with solvent, and of electrostatic bonding, respectively. According to Scheraga (1963), the quantities FE and F" in the above equation are independent of pH because they do not involve ionizable groups. The remaining parameters in the equation are highly pH dependent. For example, if the donor and acceptor groups in a side chain hydrogen bond are ionizable, then the bond will exist only in the pH range where the donor has a proton and the acceptor lacks one. Beyond this pH range, FR will be zero. These facts point out what type of effect can be expected by changing the H+ or OH- ion concentrations in the dispersed phase of a protein.

As most proteins behave as lyophilic (hydrophilic) polyelectrolyte systems, the forces of solvation and adsorption play an important part at their surface. This can be visualized with reference to electrokinetic phenomena and the amphoteric nature of the protein as follows:

+ C+ + HOH ,NH+ acid NH: alkali

A- t *, COOH PH < PI <COO--

Cationic form pI=pH (2=0)

Anionic form

where A, C, and Z. represent anion, cation, and net charge, respectively. As the net charge on a protein molecule is zero at the isoelectric point (PI) due to the formation of an inner salt (zwitterion), other biophysical properties such as the hydration, solubility, electrophoretic migration, viscosity, swelling, conduc- tivity, optical rotation, and osmotic pressure are also minimum, whereas sedimentation velocity, light scattering (Tyndall effect), surface tension, gel strength, and sensitivity to alcohol are maximum (Hartman, 1947; Jirgensons and Straumanis, 1962). Once the dipolar character of the protein is destroyed, ionic reactions (electrovalent) are created, depending upon the structure of the protein and the charge distribution. As can be visualized from the pK values and isoelectric points of different amino acids (Table IV), the ionization of different functional groups takes place in the following order: a-COOH groups of aspartic and glutamic acid residues at pH 3.6-3.8; the p-COOH group of aspartic acid at pH 4.5; the y-COOH group of glutamic acid at pH 4.6; the imidazol group of histidine at pH 6.1-6.3; a-NH, groups at pH 7.5-7.7; the SH group of cysteine at pH 9.1; the phenolic group of tyrosine at pH 9.6-9.8; the E-NH group of lysine at pH 10.4; the guanidyl group of arginine at pH above 12 (Tanford, 1962).

Being a protein, collagen is also influenced by Hf and OH- concentration. The acidic and basic functional groups govern many of the physical properties of


the collagen fiber as well as determine the reactivity of collagen to acids, bases, and ionic reagents in general. Collagen contains a fair amount of ionic groups, which in the isoelectric zone (pH 7.2) will be charged and form maximum internal salt linkages. The increase in H+ or OH- concentration breaks the internal compensation of ionic side chains and salt links, increasing the net positive or negative charge, respectively. However, the charged sites of the collagen chains do not have the freedom of action as found in the case of soluble amphoteric electrolytes (Gustavson, 1956). Hence collagen binds very small amounts of acid or alkali as compared to soluble protein in the absence of neutral salts. Nevertheless, by increasing H + or OH- concentration, the electrostatic repulsion leads to the development of a Donnan membrane potential inside the fibers (Tolman and Steam, 1918). This favors the inflow of water into the fibers. These water molecules are held either electrostatically by charged polar groups or through hydrogen bonding by polar uncharged groups and electronegative atoms. In addition, keto-imide linkage

(-C -N-), II I

occurring in every fourth residue of the collagen backbone chain and having a free carbonyl group, can function more readily as coordinator of water than regular peptide linkage

0

(-C-NH-). I1 0

During hydration the structure of the fibers is distorted, their length and diameter increase (Tolman and Steam, 1918). Sonsler et al. (1940) found that at 15% water content, protein bound about 260 molecules of water and the distance between amino acid side chains increased from 10.4 to 11.3 A, whereas at 33% water content, the spacing increased to 13 A.

Figure 13 shows the pH-dependent hydration curves of food-grade, freeze- dried collagen from cattle hide as influenced by mono-, di-, and trivalent anions. It appears that C1- and SO,2- tended to depress the hydration of collagen in the isoelectric pH range 5.0-7.0. Beyond these values, C1- increased the hydration sharply. On the contrary, pyrophosphate ions (P20,, - ) relatively increased hydration in the isoelectric zone of collagen (pH 5-7) as compared to C1- and SO,*-, but beyond pH 4 and 9, pyrophosphate did not increase hydration as C1- did. The changes in the hydration curve around pH 7.0 have been ascribed in part to the ionization of the imidazole group of histidine (Gustavson, 1956).

Two types of hydration of collagen are recognized depending on the ionic atmosphere (Gustavson, 1956). The hydration of collagen due to ionic groups and their charges in acid or base is regarded as “osmotic swelling.” However, Schut ( 1976) considered such hydration to be different from the osmotic phenomenon. On the other hand, hydration caused by the interaction of ions of

CHARACTERISTICS O F COLLAGEN IN FOOD SYSTEMS

al OI

10.0 8

F U

.= 9.5

3 2 9.0 c

c 0

E, 8.5

E, 8.0

- 0 7.5

8 % 7.0 m x f 6.5

L

al a - - C

-

+

0

8 0 - .- 6.0 s

297

.

I

5.5 111111111111 2 3 4 5 6 7 8 9 1 0 1 1 1 2

pH value

FIG. 13. Effect of mono-, di-, and polyvalent anions on the pH-dependent hydration curve of food- grade, freeze-dried collagen from cattle hide. (A) Control (HCIINaOH); (B) 0.1 M NaCl (HClI NaOH); (C) 0. I M Na2S04 (H2S04/NaOH); (D) 0.1 M Na4P207 (H3P04/NaOH). From Asghar and Henrickson (unpublished data).

neutral salts or nonionic reagents with nonionic bonds (e.g., hydrogen bond) of collagen is described as “lyotropic hydration” or “swelling.”

There are characteristic differences in the two types of swelling. Although the osmotic or electrostatic swelling results in great volume increase by dilute acid solutions, the process is reversible in contrast to the lyotropic swelling. The osmotic swelling is considered interprotofibrillar, and the integrity of the triple- helical structure of collagen remains intact. On the other hand, the lyotropic agents may alter the water structure around the collagen fibrils, interrupt the interprotofibrillar bonds and internal hydrogen bonds, or by direct binding at some sites interact with internal hydrophobic bonds. These interactions affect the interprotofibrillar structure, and hence irreversible changes may occur in the native peptide chain (Veis, 1964). However, within the fiber, the tropocollagen monomer units are not disrupted by neutral salts except that intertropocollagen bonds are influenced (Ramachandran, 1968).


2 . Effect of Weak and Strong Acids

With regard to the effect of acids on the hydration of proteins, Loeb’s (1922) valency rule implies that the same degree of swelling of protein should be produced by isovalent acids in solutions equilibrated to identical pH values. However, acetic acid produces 50% more swelling and a greater degree of peptization of collagen than HC1 does at pH 2.0. It is assumed that the weak acid (e.g., acetic acid) at pH 2.0 allows the removal of protons from the carboxyl groups of collagen (Gustavson, 1956). However, there appears to be neither marked affinity of the anions of the weak acids for the cationic groups of collagen nor any significant effect of the nonionized acid molecules at pH > 2.0. By decreasing the pH of the system to below 2.0, the fixation of molecular acid increases sharply, presumably by forming H bonds with the =CO group of peptide linkages. This acid also produces permanent swelling, which persists even after bringing the pH back to the isoelectric point (pZ). The shrinkage temperature of acetic acid-treated collagen is 12°C lower than that of untreated collagen. Some deamination of collagen also occurs by treatment with 2-3 M acetic acid (Gustavson, 1956).

Acetic acid produces both types of swelling since it involves both the electrostatic or osmotic effect and the lyotropic or Hofmeister effect (Gustavson, 1956). The latter effect in fact dominates; it is more in the nature of a specific molecular effect rather than a specific ionic effect, because it is the nonionized acid which acts as the swelling agent by competing with the peptide group involved in intermolecular linking of the protein chain. Acetic acid molecules are believed to rupture some of the hydrogen bonds and also to associate with free =CO group of the peptide linkage (Gustavson, 1956). At pH 4.5, irreversible changes also take place (Hamm, 1960).

On the addition of HCl to collagen, some H+ cations combine with -COO- anions. Thus, the net positive charge on the molecules first increases with the number of discharged carboxyl groups as a function of H + concentration. This continues until all -COO - groups have been discharged and all cationic groups are freed. As anions of monovalent strong acids do not have much attraction for the cationic groups of proteins, the anions are electrostatically compensated by the cationic groups on the protein. Consequently, the aqueous phase inside the collagen structure would contain more C1- than H + . The differential distribution of the ions between two phases causes a difference in osmotic pressure and in electrical potential. Thus, to equalize the ion concentration in two phases, water flows into collagen molecules and causes swelling (Lloyd and Shore, 1938). The helical structure, however, can withstand the strong repulsive force of the fixed charge up to a degree of ionization of 50%. At higher ionization the helix breaks down and the polypeptide stretches (Katchalsky, 1964). With further increase in H + concentration, the total positive charge on collagen becomes


constant; however, the ion (H+ and C1-) concentration in the external phase increases. Accordingly, the excess of diffusible ions in the solid phase first increases to a maximum, which coincides with the pH of maximum swelling of collagen, then declines (Procter and Wilson, 1916).

The same reasoning may also account for the fact that the swelling capacity of a dibasic acid (e.g., H,SO,) is only about half that of HC1 at identical pH value. The decline in hydration at very low pH (< 2.0) is explained by the assumption that excess anions from the added acid screen the positive charges (E-NH, + , guanidine, and imidazol groups) of the protein and reduce the interpeptide chains’ electrostatic repulsions. Gustavson (1 956) concluded that the lyotropic effects of carboxylic acids on collagen are intimately connected with the hydrogen bonding power of the molecules of the acid, and that breaking or weakening of interchain cross-links of hydrogen bonds is the main reaction leading to labilization of the chains.

It should be emphasized that views regarding the fate of the acid anions in protein solutions in the acidic pH range are divergent. The proponents of the Donnan theory assume that there should not occur any combination of anions (e.g., Cl-) with cationic centers of the protein, and all the anions are regarded as free in the internal aqueous solution without any restraint (Bolam, 1932; Wilson, 1928). The contrary view, based on thermodynamic reasonings, does not regard the internal solution as a normal aqueous phase and regards all of the anions as associated with the positively charged centers of the protein (Gilbert and Rideal, 1944). Both of these concepts seem equally applicable to the reversible fixation of the anions with the protein, but Gustavson (1956) argued that none of them apply to the irreversible fixation of the anions by cationic protein groups. For example, polymetaphosphoric acid, which probably does not interact with hydrogen bonding groups, irreversibly binds with cationic groups. However, polymetaphosphoric acid may cause some phosphorylation (probably of peptide bonds) at lower pH on prolonged treatment, and may not be implied as irreversible fixation of the anions by cationic protein groups.

3. Effect of Bases

In the case of a strong base (e.g., NaOH), the osmotic effect is pronounced and the lyotropic effect is minor, provided the treatment is not prolonged. On the other hand, the hydroxides of bivalent metals produce principally lyotropic swelling along with some osmotic effect at pH > 10, where irreversible alterations in ionic and coordinate bonds also occur. Prolonged action of Ca(OH), increases swelling, possibly due to two reactions. First, liberation of 4 0 0 - groups by divalent bases occurs (monovalent bases are less effective) (Marriott, 1933) as a result of deamidation of the amide groups of asparagine and glutamine (Bowes and Kenten, 1948a,b). Some decomposition also occurs of the guanidyl


group of arginine to ornithine and urea (Highberger and Stecker, 1941), destruction of hydroxy amino acids (serine and threonine), and hydrolysis of keto-imide linkage

(-C-N -) II I 0

between proline and hydroxyproline residues (Bowes et al . , 1953) during alkali treatment of collagen at pH 12- 14. These changes account for the displacement of the isoelectric point of alkali-treated collagen toward a lower pH (-5.0) range. With these changes cleavage of interchain cross-links in which amide groups are involved increases the probability of the uptake of cations.

Second, Ca2+ has a specific effect by forming complexes with OH- groups of hydroxy amino and residues. However, according to Lloyd and Shore (1938), the main reaction in the alkaline region (pH > 10) is the neutralization of cationic charge of the protein and electrostatic balance of the anionic groups by metal cations. In a collagen molecule the probability of getting two -COO- groups by a Ca2 + into its valency sphere is very limited. This scarcity is likely compensated by a weakly negatively charged enolized form of peptidyl bond present in the vicinity. This possibility is strengthened by the study of Carr (1953), who showed that out of 100 COOH groups of bovine serum albumin, only 8 participate in true complex formation with Ca2 + . The other 92 groups form only loose combinations of the ordinary electrostatic type.

The involvement of the enolized form of peptide bond with Ca2+ is likely to weaken the hydrogen bond. Since Ca(OH),, being a weak alkali, acts slowly, its lyotropic effect would only be observable after prolonged treatment. These changes are believed not to be a result of a Hofmeister-type molecular effect, but rather of a specific ion effect of an electrostatic nature and of the steric conditions of the collagen molecules with regard to the distance between adjacent COOH groups. Generally the swelling action of bases like NaOH and Ca(OH), follows the valency rule of Loeb (1922), provided the treatment is not prolonged. The maximum proton-accepting H + and OH- binding capacity of gelatin is about 0.9 meq/g protein at pH about 2.0 and 12.5, respectively. However, in collagen no maximum uptake of base is reached, presumably because of the guanidyl group involved in stable linkage, which requires still higher pH for its cleavage.

4. Effect of Various Salts

Before considering the effect of various salts on the hydration and stability of collagen molecules, it would be appropriate to first present a brief account of certain fundamental principles of biophysics which govern the charge and interactions among protein-ion-water systems.

As indicated earlier, a protein in solution is regarded as a hydrophilic polyelectrolyte colloidal system. Other ions, if added to such a solution, will be distributed in certain orders and hence modify the electrical properties of the


Diffusedouble layer

, Fixed (Stern) laye!

A Mobile layer \ I - , E

~. B Distance from surface Of colloid parli~le

FIG. 14. Distribution of charge in the diffuse double layer (fixed and mobile layer) in the liquid phase around the negatively charged colloidal particle (A); and variation in electrical potential (Z) at the interface with increase in distance from surface to the particle (B). E, Nernst potential; <, zeta potential.

colloid. This was first realized by Helmholz (Kruyt, 1952), who proposed that the charges around the colloids are in two layers (Fig. 14). The inner layer adjacent to the colloid surface was referred to as the “fixed” or Stem layer, and the outer layer as “mobile.” However, several workers, quoted by Kruyt (1952) and Eagland (1975b), later proved that the electrical double layer in fact exists as a “diffuse” double layer, the outer one carrying both positive and negative charges. The density of the charge in the electrical double layers decreases from the surface of colloid to the periphery exponentially rather than linearly.

The changes in charge density are expressed in terms of Nernst potential (E) and electrokinetic or zeta potential (5). The former refers to the total potential drop in charge from the surface of the colloid to the end of the diffuse layers, whereas the latter is confined to the potential drop across the mobile layer only (Hartman, 1947). Many factors can influence either the Nernst and zeta potential by changing the thickness of the electrical double layer, and hence affect the stability and hydration of the colloid particles. For example, thermal and electrostatic forces of the ions in liquid phase can change the distribution of charge in electrical double layers whose thickness is dependent on ion concentration, but independent of the nature of the ions (Jirgensons and Straumanis, 1962). Howev- er total drop in electrical potential ( E ) depends solely on the activity of ions and


H + concentration, whereas the zeta potential varies with the nature of the proteins, adsorption potential of ions at the interface, and dielectric constant of the medium (Kruyt, 1952). An increase in concentration of ions in the medium will decrease the thickness of ionic atmosphere composing the outer mobile layer of charges and hence the zeta potential. The value and the sign of zeta potential depend on the magnitude of specific adsorption of anions and cations.

According to Friberg (1976), these concepts were further elaborated by De- ryagin and Landau (1941) and Verwey and Overbeck (1948) in an attempt to explain the biophysical mechanism responsible for the stability of colloids. Ac- cording to them, two forces operate on the colloidal particles. First, the van der Waals-London attractive forces which originate in the unsaturated valency fields at the surface between two particles; they decrease with increase of distance between the particles. These forces are responsible for adsorption. Second, the Coulomb electrostatic repulsion between electrical double layers of identical signs, whose intensity is determined by the structure of the ionic layer surrounding the particles, which, in turn, is determined by the electrolytic composition of the dispersed medium. This is known as the DLVO theory after the names of those workers (Eagland, 1975b; Friberg, 1976).

Figure 15 depicts the salient feature of this theory, which suggests that if the repulsion potential ( B ) is greater than the attraction potential (A) at any distance

and the ions between lhem

form the electric double layer

which gives m e to o

j~ l lerenl f rom :he .on:~nwi

med8um o VOP der A’ms

- g z i : . “ p

-

FIG. 15. Diagrammatic presentation of the DLVO theory which proposes that colloidal stability is distance dependent on two independent potentials, that is, van der Waal’s attraction potential and the repulsion potential. From Friberg (1976). Courtesy of Marcel Dekker Inc., New York.


between the particles, the colloids will be stable. But, a low value of B , that is, an interparticle potential equal to or less than the height of energy barrier, would favor the precipitation of the particles as soon as they approach each other by the diffusion process (Friberg, 1976). However, other evidence suggests a possible involvement of hydration and solvent structure in the modification of the DLVO theory of colloid stability (Eagland, 1975b).

The ions present in the medium with the colloidal systems are also important in relation to the hydration phenomenon. The ions, if added in a colloidal solution, may be bound with the charged sites or remain free and mobile in the aqueous phase, depending upon a number of factors such as pH, nature of the ions, and nature of the colloid particles. For example, metallic ions in solution are assumed to exist as aqua-complex ions in equilibrium with their respective hydroxo complex like a weak base (Furia, 1975), as follows:

M(H2O)” + MOH(m-I)+ + H + Aqua-complex ion Hydroxo complex

(weak base)

If so, then the acid ionization constant (pK,) value of the aqua-complex ion would be a decisive factor in determining if the ion would form complexes or remain a free ion at different pH values of the medium (Basolo and Pearson, 1956). For instance, Ca2+, having a pK, value of >12.6, will bind to a negatively charged protein only under highly alkaline conditions, that is, at pH > 12.6.

Another contributing factor to hydration is the ionic radius. The highly hydrated ions with large radii are apt to remain at a distance from the oppositely charged centers on protein, whereas less hydrated small-sized ions are able to approach the charged sites more closely and hence more effectively screen the charge on the protein. The ratio of ion radius to ion charge is inversely related to the degree of ion hydration (Fennema, 1976). The polarizing power (charge/ radius or simply the electrical field) of an ion has the ability to alter the net structure of water (Fennema, 1977). On this basis, ions have been classified into two groups. First, the small ions (e.g., L i + , Na+ , OH-, HO,+) and multi- valent cations (e.g., Ca2+, Mg2+, A13+), which have strong electrical fields, are called “water structure formers” because they increase the viscosity of water by binding strongly with four to six molecules of water adjacent to them. Sec- ond, the monovalent ions of large size (e.g., K + , NH4+, C1-, I-) , on account of weak electrical fields, tend to reduce the viscosity of water. They are called “water structure breakers” (Sikorski et al., 1976; Fennema, 1976).

The effect of added ions of varying valences on oppositely charged colloidal particles is shown hypothetically in Fig. 16. It shows that the addition of uni- valent anions to the solution of positively charged colloids increases the value of the zeta potential until a maximum is reached. Accordingly, hydration may also increase due to the screening effect of the corresponding cations, which settle in a compact layer close to charged protein groups. The anions are kept farther apart


+ r

0

- r

FIG. 16. Hypothetical curves showing the effect of the addition of monovalent (curve a), divalent (curve b), and polyvalent (curve c) ions on the zeta potential of oppositely charged colloidal particles. After Hartman (1947).

from protein in the diffuse double layer. With further increase in ion concentration, the zeta potential approaches zero as the high concentration of ions com- presses the double layer around carboxyl groups, and as the repulsion charge decreases, so does the hydration. The zeta potential decreases more rapidly and approaches zero on adding divalent anions. Contrary to this, the addition of polyvalent anions reverses the sign of the zeta potential to negative, and after reaching a peak, it declines to zero with further increase in the polyvalent anion concentration (Hartman, 1947). Similar behavior may be .expected of various cations on a negatively charged colloidal system. This substantiates Loeb’s (1922) statement that polyvalent ions, of opposite sign from that of the protein, have marked influence in reversing the sign of the zeta potential and changing the isoelectric point of the protein, whereas corresponding ions (of the same charge as the protein) are unimportant.

As for other lyophilic colloids, the hydration and the stability of proteins in solution depend on two important factors. First, the electrical charge, which is due partly to the internal phase of the protein molecules and partly to the result of adsorbed ions from the medium; and second, the solvation of the molecules. Removal of these two stability factors, by changing H + content or by adsorption of other ions and dehydration agents, respectively, leads to appreciation of protein. The relative difference in the adsorption of ions in relation to colloid stability has been explained in terms of the Hardy-Schulze rule, which says that the higher the valence, the greater the coagulating effect of added ions of op-


posite charge to that of the suspended colloids. That is, the order of adsorption of ions is trivalent > bivalent > monovalent (Hartman, 1947; Jirgensons and Straumanis, 1962).

The DLVO theory presents a logical explanation for the destabilizing action of higher concentrations of the neutral salts (which do not absorb) which drops the potential faster with distance (Fig. 17). Although the total repulsion energy at very short distance is not affected, the drop in repulsion potential is appreciable at medium distance. Thus with the compression of the double layer the energy barrier disappears, leading to flocculation. The charge on the ions significantly affects the distance dependence of the potential. Consequently, electrolytes of higher charges are likely to be more effective in destabilizing emulsions (Friberg, 1976). Although this explanation supports the Hardy-Schulze rule, which states that divalent and trivalent ions, respectively, are 50-100 times more effective than monovalent ions in destabilizing an emulsion (Friberg, 1976), it does not have general applicability, because other factors such as specific characteristics of the ions, the colloidal system, and H+ concentration can offset this rule. Bull and Breese (1970) also reported disparity in the behavior of anions and cations of the same size. For example, in contrast to cations, the larger the monovalent anion, the greater is the tendency to bind to the protein and the greater is its dehydrating effect upon the protein. The flocculation power also increases with the atomic weight or ionic radii in the lyotropic series (Jirgensons and

Addition of salt changes the

The suface potential

and wth it lh

at particle touch

g l v l q Stoblllty *

FIG. 17. Hypothetical curves showing the effect of added salts (electrolytes) on the potential charge of colloids. The addition of salts may affect the maxima of the total potential at uncharged surface potential of the colloids. From Friberg (1976). Courtesy of Marcel Dekker Inc., New York.


Straumanis, 1962). Besides these considerations, certain salts of weak acids such as phosphate, carbonate, and citrate give rise to various ionic species as a function of pH of the medium. The proportion of different ionic species can be determined by the Henderson-Hasselbach equation (Jenness and Patton, 1959):

pH = pK + log saluacid.

Probably, that is why the increasing effect of salts of weak acid on hydration tends to be greater than could be expected from the valence of the anion alone according to Loeb’s (1922) suggestion.

B. SWELLING

Both electrolytes and nonelectrolytes can affect the swelling capacity and shrinkage temperature of collagen. For example, sucrose and glucose (which decrease the dielectric constant and enhance electrostatic interactions) at concentrations of about 1 and 2 M , respectively, produced maximum swelling and maximum decrease in shrinkage temperature (Lloyd and Garrod, 1948). These substances possibly influence the dielectric content of the media, which in turn affects the electrovalent linkages. High dielectric constant favors neutralization of the charged groups, whereas the low dielectric constant increases strength of electrovalent linkages and hence T, would increase. Although different factors can offset the expected effect, electrolytes have a decisive influence on the biophysical properties (swelling, solubility, gelatin, viscosity, water-binding capacity) of a protein at different ionic strengths and pH values (Hermansson, 1975; Eagland, 1975a). An increase in swelling by acids seems to be the result of the H+ ion concentration less the effect of the acid anions, and that of bases was caused by the OH- concentration less the effect of base cations (Jirgensons and Straumanis, 1962). The neutral salts at moderate concentration (1 M ) repress the swelling, with the higher concentration being more effective. It has been stated that the anions in general are more effective than cations for increasing swelling.

Anions and cations of salts can be arranged in a series in which the successive ion allows a lesser degree of swelling than the one before. The sequences usually given for different organic and inorganic ions are as follows:

Organic anions: Acetate > citrate > tartrate Inorganic anions: CNS- > I- > NO,- > Br,- > C10,- > C1- > SO,*- Inorganic cations: Li+ > Na+ > K + > NH4+ > Cs+ > Mg2+ > Ca2+ >

This broad generalization, describing the influence of salts on the properties of gels consisting of proteins or other hydrophilic colloids in water, is known as the Hofmeister or lyotropic series (Hartman, 1947; Kruyt, 1952; Kragh, 1977). The mechanism related to the existence of these series is only partly clear. The

Ba2 +


valency of the ion, however, is very important. According to Kragh (1977), the specific ion effect is also produced by adsorption, which depends on the polar- izability and size of the ions. Besides, the specific ion effect occurs in the electrical double layer at the surface since the distribution of ions in the Stem layer depends on the ionic radius. The ions on the left side of the lyotropic series may act as a hydrogen bond breaker if present in high concentration. Steinberg et al. (1960) stated that ions at the extreme left of the lyotropic series have the highest tendency to favor the disordered form (the transformation of helical to random coiled structures) of a protein.

With regard to collagen-ion interaction, two views seem to have emerged. Some workers believe in the possibility of direct ion binding to the peptide backbone of collagen (Bello et al., 1956; Mandelkem and Stewart, 1964). Von Hippel and Wong (1962) disagree with this proposition. They opine that ions affect the collagen fold indirectly by interacting with structurally bound water molecules. This conclusion was derived by treating their experimental data according to the following equation:

where K is the equilibrium constant, [SG] is the molar concentration of filled binding sites, [S] is the concentration of free salt, and [GI is the molar concentration of binding sites. By reanalyzing the earlier data according to this equation, Bello (1963) argued that ion-peptide bond interaction cannot be ruled out as an explanation of the effect of neutral salts on the collagen fold formation.

Loeb (1922) and Thomas and Foster (1925) also developed ionic series corresponding to the Hofmeister series. It appears from those series that weakly hydrated ions (e.g., CNS-) are strongly adsorbed by protein from aqueous solution, whereas strongly hydrated ions (e.g., SO,2-) exhibit little adsorption owing to their dehydration effect on protein. Thus the former have a strong swelling effect on protein at a concentration of 1 .0 M, suggesting that the effect is due to molecules of the salt rather than ions. Further, Hamm (1958) observed that salts with the same cation but different anions, as well as those with the same anion and different cations, did not function according to the lyotropic order in the case of meat protein. If so, then the Hofmeister ion series would lose some of its significance. It has also been reported that the presence of neutral salts decreases swelling in acid and alkaline media and that the anions are more active than cations. The following series indicate in descending order the swelling in acidic and alkaline media:

eP

Citrate > tartrate > phosphate > sulfate > acetate > iodide > thiocyanate > nitrate > bromide > chloride

and


However, Hamm’s (1957) studies indicated that neutral salts increased the hydration of meat proteins in the basic pH range and caused dehydration in acidic pH. He found an increased hydration effect of cations in the following lyotropic order:

Ca2+ < Mg2+ < K + < Na+ < L i t ,

while that of anions is

F- < CI- < Br- < I- < CNS

in the alkaline pH range. The anions, however, were more effective than cations in this pH range. On the acid side, anions produced a greater dehydration effect than cations. Sherman’s (1962) study supported some of these findings. Hamm (1957) explained these observations on the assumption that the positively charged groups would repel each other in the acid pH range, resulting in enlarge- ment of the interspace between peptide chains. By adding neutral salt (e.g., NaCI) the positive centers of protein bind the C1- and diminish repulsion and decrease hydration. On the alkaline side, the proteins have an excess negative charge, a part of which is involved in salt linkages with cations, particularly with the bivalent cation, which screens the charge (Hamm, 1958; Bozler, 1955). Bivalent Ca2+ and Mg2+ can also form coordinate links involving --COO-, -NH,, -SH, and - O H groups. The imidazole of histidine preferably binds to Zn2 + . Despite these generalizations, the swelling may increase in the presence of both monovalent and polyvalent ions due to their counteraction. The deamination of collagen (with nitrous acid) to replace the strongly basic E-NH, of lysine by a weakly basic OH group markedly increases the swelling capacity in the alkaline pH range (Thomas and Foster, 1926).

Interaction of Salts and Hydrogen Ions on Swelling

As mentioned before, a certain minimum concentration of H + or OH- is needed for breaking the dipolar compensation between polypeptide chains of collagen. By adding a neutral salt (1 M concentration) having a common anion of the acid, the zone of inert reactivity is eliminated, probably due to the increased concentration of the anion. This increase not only eliminates the Donnan effect, but also removes the potential barrier set up against the anions of the strong acid, which is preponderantly balanced electrostatically by, and not combined with, the cationic groups of protein. Consequently the concomitant swelling of protein is also depressed. This explains the depressing effect of NaCl on collagen hydration in the pH range 5-7 (Fig. 13).

Procter and Wilson (1916) also found that a neutral salt (e.g., NaCl) depresses the swelling of collagen in HCl by equalization of C1- distribution between internal and external phases. As the concentration of ions in the external phases


increases, the excess of diffusible ions in the internal phase decreases. In other words, the difference in anion concentration between the two phases will be reduced. Besides, the osmotic pressure of added salt would be a contributing factor. Neutral salts also screen off the polymeric charges and diminish repulsion. The stretching effect is lowered at high salt concentration since greater numbers of anions tend to combine with the cationic sites of protein due to the law of mass action, lowering the net positive charge. If so, then maximum swelling can be expected only in salt-free systems (Katchalsky, 1964). The hydration curve A in Fig. 13 demonstrates the validity of this proposition.

However, Procter-Wilson’s (1916) proposition fails to account for the fact that NaCl added to collagen in NaOH solutions did not reduce swelling, as can be seen from curve B in Fig. 13. On the other hand, CaCl, added to Ca(OH), greatly increases swelling of collagen due to lyotropic effects (Kuntzel, 1944). But, curve C in Fig. 13 shows that salt of a dibasic acid (e.g., Na,SO,) markedly suppresses the swelling of collagen (above pH 9) as compared to NaCl in NaOH solutions. The concept of the Donnan effect (Bolam, 1932) also offers no explanation for these observations. It seems that the degree of affinity of anions of the acid for cationic protein groups is important. The SO,,- have greater binding tendency than C1- with collagen. At low pH, H,SO, partly functions as a monobasic acid; this factor also favors the uptake of sulfuric acid over the HCl. All these instances suggest that the influence of neutral salts of different valency combinations or alkaline solutions containing common cations on swelling of collagen cannot be explained by any concepts which have been advanced so far (Gustavson, 1956).

Swelling of protein is an important property in foods such as processed meat, custards, and doughs, where proteins are required to imbibe and hold water without dissolving. Wettability is another functional property closely associated with hydration and swelling of proteins. It mainly depends on the hydrophilic-hydrophobic balance, the molecular surface of the protein, and the surface tension of solvent. All these characteristics determine the body and viscosity of some processed meat products (Hermansson and Akesson, 1975; Kinsella, 1979). In this regard the actomyosin and other myofibrillar proteins are thought to have the sole role (Hamm, 1957; Nakayama and Sato, 1971a,b,c). Little consideration has been given to collagen, although it seems to have a greater potentiality for water binding than other proteins. This aspect needs to be ascertained.

C. EMULSIFYING CAPACITY

As indicated earlier, proteins are important functionally in various food bio- systems. They have the potentiality to form and stabilize oil/water emulsions in a number of meat and nonmeat products (Wolf and Cowan, 1971; Friberg, 1976),

3 10 A. ASGHAR AND R. L. HENRICKSON

which otherwise, in the absence of an emulsifier, are unstable on account of the positive free energy resulting from interfacial tension. The surface characteristics of a protein are related to the emulsifying capacity and emulsion stability in the product. The charged protein molecules encapsulate the fat globules and lower the interfacial energy between oil/water phases by mutual repulsion, and hence prevent coalescence of fat droplets in the emulsified products (M. J . Y. Lin et al., 1974; Hermansson and Akesson, 1975). The DLVO theory, already mentioned in Section VI,A, well explains the overall mechanism of emulsion formation and stability. Hence, all those factors which can affect the electrical double layer will influence the stability of the emulsion.

The opinions of different workers, however, are divergent as to the state of a protein which would result in maximum emulsion capacity. According to Kamat et al. (1978), denatured proteins provide greater emulsion stability than native ones, and optimum performance can be expected at or near their isoelectric point, where the protein adsorption and viscoelasticity at the oillwater interface is maximum and repulsion is minimum. It is assumed that proteins at their isoelectric point stabilize emulsions by a mechanism of adsorption and interfacial denaturation to produce a physical barrier (steric) against coalescence of dispersed phase (Cante et al., 1979). Trumbetas et al. (1979) also showed maximum nonpolar interactions between protein and fat on emulsification at the isoelectric point of the protein (protein solubility at this point is expected to be minimum). These findings support the view of Boyer et al. (1946), who indicated the existence of nonpolar interactions between protein and oil phases, whereas carboxylate groups (in the case of free fatty acids) play only a minor role (Spector, 1975).

The chemical forces which confer stability to protein-lipid complexes depend largely on lipid composition. For example, fatty acids can form ester or amide linkages by involving a COOH group with OH or NH, groups of a protein, respectively (Burley, 197 1). Theoretically, ionic linkages are also possible between phosphate groups of phospholipid and appropriate charged groups (-NH,+) of proteins. The groups having dipole character can also be expected to interact with ionic groups (Krimm, 1968). However, neutral triglycerides can react with protein only by various weak van der Waals forces, which are composed mainly of London or dispersion forces (Salem, 1962). Besides, the hydro- carbon side chains of the protein are assumed to be in the nonpolar interior of the molecule in aqueous media, where the interactions between nonpolar groups of protein and lipid are favored by the necessity to avoid the surrounding water. Such interactions created by the presence of water between nonpolar groups are known as hydrophobic bonds (Burley, 1971), which probably are mainly responsible for conferring stability to protein-lipid complexes. The strength of these bonds increases with rise in temperature up to 5OoC, whereas the strength of most other bonds decreases (NCmethy, 1967). The stability of ionic bonds is also


dependent on the dielectric constant of the medium, and for aqueous media, on the ionic strength and pH as well.

It may, however, be emphasized that the surface activity of a protein is a function of the ease with which it can unfold, rearrange, and adsorb at an interface. Since the solubility in aqueous phase may change the surface activity, it is not surprising that a number of studies have found a positive relationship between emulsifying capacity of proteins and their solubility (Pearson et al., 1965; Saffle, 1968; Yatsumatsu et al., 1972; Hagerdal and Lofquist, 1978; Pearce and Kinsella, 1978). Other workers also found unstable emulsions at isoelectric points of the protein (Franzen and Kinsella, 1976; Aoki et al., 1980). Similarly, Crenwelge et al. (1974) noted increased emulsifying capacity as the pH of the emulsion diverged from the isoelectric region of the protein. It must be realized that the solubility of protein is a relative property, since all proteins can be solubilized by using appropriate dispersing media. Hence it would be more appropriate to express the emulsion stability in more appropriate terms, such as pH, ionic strength, and protein concentration, rather than solubility. Very recently, Holm and Eriksen (1980) reported that the emulsifying capacity of protein solution increased significantly on removing the low-molecular-weight components by dialysis.

The emulsifying capacity of proteins of animal origin, such as myosin, actin, actomyosin, tropomyosin, and sarcoplasmic proteins, have been studied extensively (Fukazawa et al., 1961; Hegarty et al., 1963; Carpenter and Saffle, 1965; Swift, 1965; Maurer et al . , 1969; Neelakantan and Froning, 1971; Dawood, 1980). Saffle (1968) and Schut (1976) have reviewed this issue in detail, but little has been reported about the emulsifying capacity of collagen. Being an insoluble protein, collagen may be expected to be of little significance as an emulsifier. However, properly processed and modified food-grade collagen derived from hide may prove a better emulsifier than nonfat dry milk (Satterlee et al., 1973). On the basis of extensive study on protein emulsions, Smith et al. (1973) proposed that very small particles (finely divided protein) can aid emulsion stabilization. Collagen derived from hide can easily conform to these characteristics. Beside this, better results may be expected if a hydrophilic-hydrophobic balance (HHB) of collagen and fatty constituents may be established as proposed by Griffin (1 949). The appropriate values of HHB have been reported for other oil and surfactant and protein systems (Becher, 1965; van Eerd, 1971). Despite certain limitations (Boyd et al., 1972), the HHB value provides a useful approximation for making stable emulsions.

D. FOAMING

The ability of a protein to form a stable foam is one of the great functional characteristics in food science, especially in baking technology. Unlike an emul-


sion, the discontinuous phase in foam is the gas droplets, encapsulated by an aqueous film of protein, which lowers the interfacial tension between the gas and water phases (Kinsella, 1979). The presence of lipid destabilizes the protein film and hence is detrimental to foaming (Yatsumatsu et a / . , 1972). Foaming is also affected by pH, protein concentration, temperature (Eldridge et al., 1963), and partial proteolysis (Horiuchi et al., 1978). The latter authors have associated the foam stability with surface hydrophobicity of a protein molecule. The whipping property of a protein is closely related to the foaming capacity. However, no information seems to be available regarding the foaming capacity ,of food-grade collagen derived from hides.

E. VISCOELASTICITY

The unique physicochemical properties of fibrous collagen have been utilized in the fabrication of useful products such as edible collagen sausage casings (Braun and Braun, 1956; Reissmann and Nichols, 1960; Cohen, 1964; Talty, 1969; Kidney, 1970). The making of sausage casing depends on the viscoelastic characteristics of the dispersed collagen. Due to these characteristics, collagen dispersions can serve as a binder and a lubricant.

A number of studies have reported the viscometric characteristics of soluble collagen (Runkel and Lange, 1937; Kahn and Witnauer, 1966; Cerny et al., 1970; Whitmore et al., 1972). It has been shown that satisfactory cold dispersion of collagen cannot be made in the pH range 4.3-8.5 without using an additive like guar gum, which reduces reaggregation of unswollen fibrils (Whitmore et al., 1972). The cold acid dispersion at low ionic strength (< 1 .O) consists entirely of swollen fibrils at pH < 4.3 (Borasky, 1963) and temperature below 50°C.

VII. NUTRITIONAL ASPECTS OF COLLAGEN

While the functional properties of proteins can influence the aesthetic value and organoleptic characteristics of different processed food products, the digestibility, biological availability, and relative proportion of the essential amino acids (Table VI) for optimum utilization of nonessential amino acids, determine the nutritional value of the proteins. These variables affect the efficiency of potential biological utilization of dietary proteins for meeting various requirements of the human body (Morrison and McLaughlan, 1972). Physiological availability of amino acids of a protein is also influenced by various processing treatments. For better appreciation of the subsequent discussion on the nutritional aspects of collagen, first a brief explanation of the relevant terminology and biological assaying of protein quality seems appropriate.


TABLE VI ESSENTIAL AMINO ACIDS FOR GROWTH AND MAINTENANCE OF HUMAN SUBJECTS

AND RATS

For growth For growth For maintenance Amino acid in rats in children of adults

1. Isoleucine 2. Leucine 3 . Lysine

X

X

X

X

X

X

X

X

X

4. Methioninea X X X

6 . Threnonine X X X

8. Valine X X X

9. Histidine X X Not essential 10. Arginine X ? Not essential

"Cystine and tyrosine content can spare the need of methionine and phenylalanine, respectively, up to 30-50%. Arginine is also essential for birds. Human subjects can synthesize arginine, but the rate may be limited, hence dietary supply may be needed for optimum growth. Glycine is essential only for birds.

5 . Phenylalanine" X X X

7. Tryptophan X X X

A. PROTEIN QUALITY ASSAYS

A number of biological- and chemical-based procedures have been described to evaluate the nutritional quality of food proteins. A detailed discussion of those methods can be found in a number of comprehensive reviews (McLaughlin, 1974a,b; Bender, 1975; Pike and Brown, 1975; Bodwell, 1977a,b; Hackler, 1977a,b; Sammonds and Hegsted, 1977). They include nitrogen balance studies with human subjects, growth studies with laboratory animals (generally rats), chemical assays, bioavailability of individual essential amino acids, and other bioassays (Bodwell, 1977b). These methods can also be classified as primary (direct) and secondary (indirect) assays. Another classification could be one- point assays and multipoint assays (Lachance et al., 1977).

The primary assays of protein quality of different foods are directly performed on human subjects for a specific period (35-45 days) to determine the nitrogen balance index. The biological value or net protein utilization can be derived by direct comparison of different food proteins involving human subjects for 12-15 days. The multipoint nitrogen balance index (NBI) assay can be performed in 10 days using adult human subjects (Lachance et al., 1977).

For routine and quality control purposes, indirect assays of protein quality are generally performed by using either laboratory animals, mostly rats (Hackler, 1977) or microorganisms, such as Leuconostoc mesenteroidis, Streptococcus

faecalis (Horn and Warren, 1961; Horn et al., 1954; Teeri et al., 1956), Strep-


tococcus zymogenes, protozoa Tetrahymena pyriformis or Tetrahymena ther- mophila WH,, (Evancho et al., 1977; Satterlee et al., 1977, 1979). Proteolytic enzymes such as papain (Buchanan and Byers, 1969) and the pepsin-pancreatin system (Akeson and Stahmann, 1964) were employed to study the digestibility of the protein in vitro and to correlate them with in vivo digestibility. Further improvement in the enzymic method has been achieved by using multienzyme systems such as pepsin-trypsin-pancreatin and trypsin-chymotrypsin-peptidase (Hsu et al., 1977).

The data on nitrogen content of diet, carcass, feces, urine (including endogenous and metabolic nitrogen), growth, protein consumed, amino acid content of protein, etc. are collected depending upon the nature of the experiment. The protein quality is then expressed in any one or more of the following terms depending upon the experimental conditions.

I . Digestibility

The digestibility of a protein is the primary determinant of the availability of its amino acids. It is defined as the proportion of food which is absorbed, that is:

Digestibility = x 100

If the correction for metabolic loss in feces is not made, the value is called an ‘‘apparent digestibility.”

N in diet - N in feces - N in metabolism N in diet

2. Biological Value

Biological value (BV) is a single-point assay, based on nitrogen balance. This indicates the proportion of absorbed nitrogen which is retained in the body for maintenance and/or growth (Mitchell, 1924) that is:

N in diet - (N in feces - N metabolic) - (N in urine - N endogenous) N in diet - (N in feces - N metabolic) BV = x 100.

However, if the correction for metabolic and endogenous losses is not made, the value is termed ‘‘apparent biological value. ’ ’

3 . Protein Efficiency Ratio

Protein efficiency ratio (PER) is defined as the ratio of weight gain to protein consumed (Osbome et al., 1919) in a specific period (e.g., 28 days), using a single level of protein (10%) in the diet:

Weight gain by test animal Weight of protein consumed PER =


This one-point assay is the AOAC’s approved method for determining the nutritive value of proteins, even though it yields variable results (Bender and Doell, 1957) and has some limitations (Jacquot and Peret, 1972; Satterlee et al., 1979). There is now an overwhelming consensus that serious problems arise with PER as a protein quality indicator. This was reflected in a symposium2 on protein efficiency.

4. Net Protein Ratio

The principal drawback in the assay of PER was removed by Bender and Doell (1957) by measuring the net protein ratio (NPR) value of the diet. This was achieved by including a second group of experimental animals, using a protein- free diet. It was based on the assumption that protein required for preventing weight loss of the protein-free group would be an estimate of the maintenance requirement of the test animals. This two-point assay needs only 10-14 days (McLaughlan, 1974) and NPR is derived from the following expression:

Weight gain + weight loss by protein-free group Protein consumed NPR =

5. Net Protein Utilization

Earlier Bender and Miller (1953) also suggested the estimation of net protein utilization (NPU) as a measure of protein quality. This single-point assay is identical to NPR, except that the amount of carcass nitrogen is involved in the estimate instead of the body weight of the test animals, as follows:

Body N - body N of protein-free group N consumed NPU =

McLaughlan (1974b) believes that NPR is essentially equal to PER + I . 5 , whereas NPU is equal to BV X protein digestibility.

6. Relative Nutritive Value

The relative nutritive value (RNV) provides an estimate of the protein quality by feeding the protein in question to test animals at three levels for about 3 weeks, including a zero protein level (Hegsted et al., 1968). A comparison is then made using lactalbumin as control with the regression lines of body weight to protein intake. The RNV gives values almost identical to the NPR assay (McLaughlan and Keith, 1975).

*Overview of outstanding symposium in Food Science and Technology, Food Technol. (Chicago) 31, 69-93 (1977).


A Weight change on diet bal

N growth

ratio

Net protein ratio

0 10% protein Protein or N intake

B

N i c e

(standard) Bioloaical

N intake

FIG. 18. proteins. From Allison (1964).

Relationship among different bioassay methods for determining the nutritive value of

7 . Relative Protein Value

The relative protein value (RPV) is a simplified version of the RNV assay, in which the zero protein level is deleted in the procedure (Hegsted and Chang, 1965). Lactalbumin is still used as a control. Consequently, the regression is derived in the region at which protein intake and body weight exhibits a linear relationship. However, the RPV assay is highly influenced by lysine and threonine content of a protein (Hackler, 1977b). For example, lysine-poor proteins tend to give low slopes, which underestimate protein quality of such proteins. Threonine-deficient proteins give steep slopes and tend to overestimate protein quality of these proteins. McLaughlan (1976) has suggested another method for evaluating protein quality, called “relative nitrogen utilization. ” Figure 18 depicts the salient differences among some of these bioassays of protein quality including the nitrogen balance index (Allison, 1964). Very recently Heger and Frydrych (1980) have reviewed the merits of each of the above- mentioned bioassays of protein quality, whereas Evans and Witty (1980) have elaborated the merits and demerits of using protozoa for determining the protein quality of foodstuffs.

8. Chemical (Amino Acid) Score Assays

These assays of protein quality are based on the comparison of amino acid profiles of a protein with whole egg protein, assuming that all of the amino acids are 100% available in vivo, which in fact is not the case (Bender, 1973). The score can be computed, using the following expression:

mg of amino acid in 1 g of test protein mg of amino acid in 1 g of reference protein Amino acid score =


Mitchell and Block (1946) used the percentage of the most limiting essential amino acid, based on Liebig’s law of minima, to evaluate protein quality by comparing it with the essential amino acid profile of the whole egg, and designed this percentage as the “chemical score” to equate it with biological value. Oser (1951) considered the total essential amino acids as a better indication of protein quality. The geometric mean percentage of the percentages of all essential amino acids (by comparing with those of whole egg) was named “essential amino acid index” (EAA index). As Oser’s method of calculation differs from that of Mitchell and Block, it gives different results especially with low-quality proteins. However, Kofranyi (1972) remarked that none of the methods correctly interprets the experimental results, since neither takes into account the role of nonessential amino acids. The procedure of McLaughlan and Keith (1975) which involves three amino acids, namely, lysine, methionine, and cysteine, for computing the chemical score may also suffer from the same limitations.

A number of research workers attempted to predict PER of the protein from its essential amino acid content. Satterlee et al. (1977) predicted the PER by expressing the essential amino acid profile of the test protein as a percentage of a reference casein’s essential amino acid profile. On the basis of extensive trials on the PER assay and amino acid analysis of different products, Alsmeyer et al. (1974) derived the following regression equations for computing PER from amino acids data:

(1) (2)

(3)

Equations (1) and (2) are applicable to meat products, whereas Eq. (3) relates to meat-containing cereals, yeast, etc. (Happich et al., 1975).

Apart from these, a number of other assays have also been suggested for measuring the nutritional quality of protein. They include assays based on reple- tion of exhausted nitrogen stores and measurement of plasma-free amino acids (Hartog and Pol, 1972), clinical methods (Scrimshaw and Young, 1972), various modifications of nitrogen-balance tests, the PER and related methods (Jacquot and Peret, 1972; Hartog and Pol, 1972), protein utilization (Kofranyi, 1972; Payne, 1972), bioavailability of amino acids (Gupta et al., 1958; Carpenter et al., 1972), and chemical estimations of certain essential amino acids (Pongpaeu and Guggengeim, 1968; Momson and McLaughlin, 1972; Carpenter and Booth, 1973; Finley and Friedman, 1973; Lakin, 1973; Holsinger and Posati, 1975; Hurrell et al., 1979).

Bodwell (1977b), while reviewing the various approaches of protein quality evaluation, concluded that, although the values obtained from rat assays are related to the nutritional value of proteins in general, their significance in terms

PER = -0.684 + 0.456 (leucine) - 0.047 (proline), PER = -0.468 + 0.454 (leucine) - 0.105 (tryptophan),

+ 0.21 1 (histidine) - 0.944 (tryptophan). PER = -1.816 + 0.435 (methionine) + 0.780 (leucine)


TABLE VII FAOiWHO STANDARD FOR ESSENTIAL AMINO ACID REQUIREMENTS FOR ADULT

HUMAN SUBJECTS

Amount (gi100 g protein) Essential amino acids

I . Leucine 2. Phenylalanine-tyrosine 3 . Lysine 4. Valine 5 . Isoleucine 6. Threonine 7. Methionine-cystine 8. Tryptophan

7.0 6.0 5.5 5.0 4.0 4.0 3.5 1 .O

of protein for humans is not well defined. Moreover, in those cases where a comparative study has been made involving the same samples of proteins with both human and rat (Bodwell, 1977c), no consistent relationships were found between the bioassays of nutritive value in human or rat.

Since 1935, different international committees have made a number of recom- mendations regarding the protein and amino acid requirements of human subjects (KofrBnyi, 1972). In 1965, the F A 0 expert committee on protein requirements recommended a provisional reference, keeping in view the human need for essential amino acids. As it contained excessive amounts of tryptophan and sulfur-containing amino acids, another FAO/WHO Expert Committee suggested a revised standard reference in 1973 (Table VII).

It may be pointed out that all of the nonessential amino acids are metabolically glucogenic in a reversible manner, that is, they are interchangeable with each other and with certain energy-yielding carbohydrates. Among the essential amino acids, only a few (arginine, isoleucine, threonine, valine, cysteine) are glucogenic but nonreversibly, others (leucine, phenalalanine, tyrosine, isoleucine) are irreversibly ketogenic. The metabolic fate of a small group of essential amino acids (lysine, histidine, methionine, tryptophan), which are invariably found to be limiting in most food systems, is uncertain (Payne, 1972).

B. DIGESTIBILITY OF COLLAGEN

The nature of catabolic changes in collagen has been discussed in Section IV,B, indicating that native collagen is almost resistant to proteolytic attack, hence is regarded as indigestible. However, denatured collagen can be acted upon by a number of proteolytic enzymes. Cooking and highly acidic conditions in the stomach (pH < 2.0) cause denaturation and unfolding of the triple helix of collagen, making it susceptible to enzymic digestion. Mandl (1961) has reviewed


the effects of temperature, acids, swelling, liming, and particle size on the susceptibility of collagen to trypsin attack, even though the extent of cross- linkages and steric structure do influence the digestibility (Banga and Balo, 1965).

Nutritional trials with rats by Whitmore et al. (1975) have indicated that the apparent digestibility of fibrous collagen was 90%, and that 1 g of collagen was equivalent to 1.5 g of gelatin as an energy source. Moreover, pathological examination of body organs (stomach, intestine, heart, trachea, larynx, lungs, pancreas, liver, kidneys, urinary bladder, spleen, pituitary, thyroid, parathyroid, thymus, brain, adrenals, testes or ovaries, and uterus), revealed no abnormal lesions on feeding the rats with a diet containing up to 20% collagen for 90 days. However, the kidneys of rats fed collagen weighed more than those of rats fed casein. The functional hypertrophy of the kidney was thought to be due to the high nitrogen content of the collagen (18.6% N of collagen vs 15.9% N of casein). The hypertrophy of the kidneys due to high nitrogen content in the diet has been reported by Osborne et al. (1926-1927). The very low nutritional value of gelatin, a derivative of collagen, has been proven by the studies of Chapman et al. (1959) and Rama-Rao et al. (1964).

C. BIOLOGICAL VALUE AND PER OF COLLAGEN

Mitchell and Carman (1926) stated that different meat cuts may be equal in digestibility, but may vary in biological value due to a difference in connective tissue content. In another study, Mitchell et al. (1927) reported that a cut of veal, evidently fibrous, had a BV of only 62, whereas similar beef cuts had a BV of 69. Similarly, DvofAk and Vognarova (1969) obtained a low chemical score from amino acid analysis of cuts rich in connective tissue. Meat from steers was found to have a lower BV than meat from yearling bulls, and Avshalumova (1974) attributed this finding to the content of collagen. The study of Bender and Zia (1976) also provided supporting evidence for Mitchell and Carman’s contention that low-quality meat (shin) with 23.6% connective tissue had an NPU of 69, whereas high-quality meat (filet beef steak) containing 2.5% collagen had an NPU of 82. The NPU of meat generally varies from 62 to 78, the average being 74 (Food and Agriculture Organization, 1968). The NPU of meat decreases on cooking mainly due to the loss of available methionine in the presence of other foodstuffs (Bender and Husaini, 1976). Lee et al. (1978) found a highly significant negative correlation of meat collagen content with PER ( r = -0.98) and essential amino acids content (r = -0.99, p < 0.001).

The amino acids composition of collagen has been discussed in Section II1,A. It can be seen that among the essential amino acids, methionine, lysine, and threonine are limiting in native collagen, whereas tryptophan is practically absent. The total indispensible amino acids constitute 26.3-28.4% [al(I), 27.4%; a2(I), 28.4%; al(II), 26.6%; al(III), 26.3%]. The nonessential amino


acids constitute 71.6-73.7% of the collagen molecules. The bioassay of gelatin by Rama-Rao et al. (1964) has shown that all of the essential amino acids, except arginine, are limiting as compared to the required pattern.

These facts suggest that the addition of collagen to formulated meat, to produce high-priced products by restructuring, is likely to lower the nutritive value of the resulting products. This apprehension probably inspired the USDA to propose interim regulations on the nutritional quality of such meat products. Accordingly, a minimum PER of 2.5 and a minimum essential amino acid content of 32% are specified for most of the fabricated products (U.S. Depart- ment of Agriculture, 1981). However, Bodwell (1977b) considered that in the use of such quantity-quality breakpoints where PER <2.5, 65 g of protein are required to supply 100% of the maximum value per serving. A PER 2 2.5, requiring 45 g of protein or less, is scientifically illogical and economically

By using varying amounts of collagen in the meat product bioassay, Lee et al. (1978) computed the following linear regression equations to estimate the PER of meat products from their collagen or essential amino acid content:

costly.

PER = -0.0229(collagen %) - 3.1528, PER = -0.0632(ten EAA %) - 0.1539, PER = -0.08084(seven EAA %) - 0,1094.

According to these regression equations, a PER of 2.5 corresponds to 28.5% of collagen in the test products, or 32.2% of the amount of seven essential amino acids (isoleucine, leucine, lysine, methionine, phenylalanine, threonine, and valine) or 42% of the amount of ten essential amino acids. This suggests that there will not be any risk of a low PER or imbalance of essential amino acids in the product if the collagen content in the product is less than 28.5%. The earlier finding by Ashley and Fisher (1966) that chicks fed on a. 10% gelatin + 3% casein diet had weight gains equal to those fed on a 13% soy protein + 0.2% methionine diet provides further support for this contention. Similarly Er- bersdobler et al. (1970) have found some improvement over the control group in daily gain and feed conversion in male rats by incorporating collagen or gelatin at levels up to 5% of the diet weight. Hence, collagen, if used with balanced protein and within limits, should not decrease the nutritive value of the diet. However, a collagen content more than 28.5% in the diet is likely to lower the PER to below 2.5. Only in such situations would the problem of fortification arise.

D. POSSIBLE FORTIFICATION METHODS OF COLLAGEN

A most convenient way of improving the nutritional value of proteins in a food system is by fortification directly with the required content of the limiting amino


acids. Despite simplicity, this approach has certain inherent drawbacks. Some amino acids, if added in free form, may change the taste and flavor of the product (Beigler, 1969; Yamashita et al., 1970; Klaui, 1974). For instance, methional and dimethyl disulfide formed from methionine in a processed food give an undesirable flavor (Hippe and Warthesen, 1978). Free amino acids are susceptible to Strecker degradation and Maillard-type reactions with

0 II

-C or -CHO

groups of reducing sugars giving a brown coloration (Reynolds, 1963, 1965; Eskin et al., 1971). The are likely to leach out with the aqueous phase during processing (Bressani, 1975; Fujimaki et al., 1977). The addition of free amino acids may also have nutritional implications. A number of studies have shown a difference in the metabolic efficiency with which free amino acids and those of proteins are transported across the intestinal wall. Small peptides are reported to be more readily assimilated than free amino acids (Craft et al., 1968; Matthews et al., 1968; Adibi, 1971; Cheng et al., 1971; Lis et al., 1972; Burston et al., 1972).

These problems could be resolved by covalently linking the limiting amino acids either by chemical or enzymatic reaction with the protein to improve its nutritional value (Feeney and Whitaker, 1977). Sheehan and Hess (1955) first suggested the use of carbodiimides as the coupling reagents for peptide synthesis. Based on a similar approach, Bjarnasson-Baumann et al. (1977) achieved improvement in the nutritional assay of whey protein by covalent incorporation of phenylalanine, tyrosine, methionine, and isoleucine. Li-Chan et al. (1979) reported on the fortification of wheat gluten with lysine and Voutsinas and Nakai (1979) enriched soy protein with methionine and tryptophan using similar methods. The casein was also enriched by a similar approach (Puigserver et al., 1979).

Fortification of proteins with limiting amino acids has also been achieved enzymically by the plastein reaction (Yamashita et al., 1970, 1975, 1976; Lalasidis and Sjoberg, 1978). Detailed information on this aspect is available in a review by Fujimaki et al. (1977). Briefly, the process requires two enzymic reactions, viz. protein hydrolysis followed by re-synthesis. However, Yamashita et al. (1979a) modified the original procedure into a one-step process, which has been applied to soy protein (Yamashita et al., 1979b).

Although covalent binding of limiting amino acids with proteins seems chemically quite feasible, some biochemical complications can arise with these approaches. For example, prior to fortification, hydrolyzing gluten with mild acid also caused deamidation of asparagine and glutamine (Wu et al., 1976), and subsequent enrichment of the hydrolysate with diamino acids such as lysine is


likely to result in different isopeptide bonds such as a-E, y-a, y-E, p-a, and p-E isopeptide linkages. Some of these isopeptide bonds may not all be readily digestible (Kornguth et al., 1963) even though Mauron (1972, 1973) has stated that E-(y-glutamy1)-L-lysine and E-(a-glutamyl-L-lysine are available as a lysine source to rats. However, isopeptide bond formation can be minimized by using E-NH, group-protected lysine (NE-benzylidenelysine) in the reaction (Li-Chan et al., 1979; Li-Chan and Nakai, 1980). Moreover, N&-benzylidenelysine is reported to be almost 100% utilized as a source of lysine (Finot et al., 1977a), including Schiff base and other lysine derivatives (Finot et al . , 1977b). Puig- server et al. (1979) also did not find any adverse effect of feeding chemically enriched casein to rats on plasma amino acid pattern and PER, although their in vitro observations did show lower digestibility of the enriched casein than that of the control.

One important requirement in covalent binding of desired amino acids to a protein is that the amino group taking part in the formation of a peptide bond must be nonionized (Snellman, 1965). Empirically this means that the pH of the medium should be about 8.0 or less, if one is to distinguish between the a-NH, group and the E-NH, group of lysine. The E-NH, is essentially all ionized at pH 7.6 (pK, = 10.0), hence it will not be able to react. However, in peptide chains, containing three or more amino acid residues, the pK value of the a-NH, group is in the range 7.6-7.8 and, therefore, such amino groups can react rapidly.

All of these findings suggest that covalent binding of limiting amino acids to collagen may also be possible, however, as yet no experimental attempts have been reported. Even though success may be achieved in the laboratory, the commercial feasibility of such approaches appears to be questionable in view of the economics of the process. It is also not known how such chemical enrichment of the proteins with limiting amino acids would affect the functionality of the resulting proteins.

VIII. FOOD USES OF COLLAGEN

A. PRODUCTION OF EDIBLE FIBROUS COLLAGEN

Animal skins contain the bulk deposit of collagen (Section 11,A); thus, by- products of the tannery can be utilized in the commercial production of edible fibrous collagen. Montagna et al. (1970) and Mier and Cotton (1976) have provided extensive information on the structure and composition of skin from a biological viewpoint. However, Fig. 19 presents the structural features of skin and other layers diagrammatically as applied by the leather industry. The outer layer (epidermis) is usually removed along with hairs and hairroots (keratin, rich in sulfur amino acids) during preparatory operations. This layer has been of little


Grain

Grainamurn iunctton

Corium

Flesh

\

Epidermis

Sebaceous gland Erector pill muscle

Hair root

Artery

Vein

Colbgen fibers

lnterfibrillar material

Fat

1

FIG. 19. components. From Johns (1977).

Schematic diagram of the bovine hide cross section, showing various layers and different

TABLE VIII CHEMICAL COMPOSITION OF SKIN

Constituents Amount

1 . Water 2. Protein"

a. Globular serum protein b. Glycoproteinb c. Collagen

i. Neutral salt-solubleO ii. Acid-soluble0

3. Mucopolysaccharidesc a. Hyaluronic acid b. Dermatan sulfate c . Heparin d. Condroitin 4-sulfate e . Condroitin 6-sulfate f . Heparitin

4. Nucleic acidsd a. Ribonucleic acid b. Deoxyribonucleic acid

5. Inorganic matter

6@65% 3@35%

0.5-0.7% 0.08% 9@95%

0.03-0.6% 0.05-2.6%

0.345% 525 mg/g dry skin 205 mgig dry skin 71 mgig dry skin 69 mg/g dry skin 68 mg/g dry skin 58 mg/g dry skin

1.0% 0.8% 0.2% 0.8%

"Bowes et al. (1957). bYates (1968); Veis et al. (1960) <Kofoed and Bozzini (1969). dBose (1963).


commercial importance. The “grain” layers are relatively rich in blood vessels and muscle but are low in collagen content as compared to the “corium” layers, which are mostly composed of collagen (Bowes and Raistrick, 1968) with small amounts of elastin, reticulin, fibroblasts, and some globular serum proteins (Humphrey et al., 1956, 1957; Cooper and Johnson, 1958; Cooper ef al., 1967; Mellon et al., 1960). The inner subcutaneous layer is rich in fatty components. Recently Tajima and Nagai (1980) have shown that the distribution of collagen in different layers of skin also varies significantly.

Table VIII, derived from various sources, summarizes the overall composition of skin. It shows that, besides water, collagen is the principal protein, whereas hyaluronic acid and dermatan sulfate are the main mucopolysaccharides in skin. Other mucopolysaccharides are present only in traces (Meyer et al., 1957; Schiller, 1966; Cifonelli and Roden, 1968; Kofoed and Bozzini, 1959; Barker et al., 1969). Whether or not cattle hide contains all the minor mucopolysaccharides is not certain. The lyophobic fraction in the skin consists of triglycerides, waxes, sterol esters, and free fatty acids, with phospholipids being absent (Nicolaides et al., 1968; Wilkinson, 1969).

For processing of leather, the lime-treated hides are split in two layers, that is,

TABLE IX SALIENT FEATURES OF FIVE COLLAGEN PRODUCTS

Flow rate (1bihr)b Moisture Gelatin0

Processing Particle contentb (7% dry On wet On solid Product machine“ descriptiono (7%) wt. basis) basis basis

Feed - (limed splits)

Product 1 Comitrol, 0.06 in.

Product 2 Comitrol, microcut

Product 3 Disc mill

Product 4 Comitrol, 0.2 in.

Product 5 Disc mill

- 76.0 - 2030 487.2

Relatively 78.2 2.38 2200 479.6 large, densely matted fibers

small, less dense with bluish coat

bundles

vidual fibers

vidual fibers

Relatively 85.5 3.06 3300 478.5

Separated fiber 83.2 2.79 2860 480.5

Shorter indi- 82.9 1.17 2800 478.8

Shorter indi- 86.7 5.91 3600 478.8

<‘From Komanowsky er al. (1974) bFrom Turkot et al. (1978).


A

LIMED SPLITS

STRIP

K N I F E

0.1% EENZOIC A C I D SOLUTION

POINT (DH 5.3)

PR?OUCT PRCOUCT

PRCDUCT PRODUCT 2 -4

B

DRAINING SCREEN

SPLITS (TWO)

ACID MIXING TANK \

STORAGE W A T E R 7 HOPPER \

325

FIG. 20. Flowsheet for the production of food-grade fibrous collagen from cattle hides. (A) Pilot- plant production; (B) commercial production. From Turkot et al. (1978). Courtesy of Institute of Food Technologists, Illinois.


an outer “grain” and inner “flesh” part, with the help of a machine. The “grain splits” are meant for leather production, whereas the “flesh splits” are used for making suede leather and sausage casings (Talty, 1969; Kidney, 1970) and for the production of food-grade fibrous collagen. About 30% of over 45 million hides annually produced in the United States, not suitable for leather-making for various reasons, may represent a low-cost source of fibrous collagen (Whitmore et al., 1972; Komanowsky et al., 1974).

However, special processing technology is needed for the production of food- grade fibrous collagen with strict control on denaturation and microbial con- tamination of collagen so that it could be used in various food systems. A breakthrough in this respect was made at the Eastern Regional Research Center, Philadelphia (Elias et al., 1970; Whitmore et al., 1970), and finally Komanowsky et al. (1974) developed a process which yields five types of food- grade collagen to meet these requirements. Table IX shows the important characteristics of the five types of food-grade collagen products.

The flowsheets in Fig. 20 present the salient features of the overall process. Briefly, the flesh splits are fed to strip cutters, and the resulting pieces move onto a rotary cutter which reduces them to about %-in. particle size. By means of a conveyor, the collagen particles are transferred to a hide processor containing water, propionic acid, and benzoic acid (1000:3: 1 w/w/w), tumbled there for 4 hr, and then drained on a screen conveyor. The processed pieces are fed to either a comitrol or disc mill by cavity pumps, and then to the microcut depending on the type of desired product. In all cases, the temperature is reduced to 1.7”C before packing, and the products are stored at - 18°C to keep them micro- biologically safe for subsequent food uses.

B. PRODUCTION OF MICROCRYSTALLINE COLLAGEN

Another fibrous form of collagen which has been made from hide for various uses is microcrystalline collagen. The preparation procedure for this product has not been published for proprietary reasons. However, Battista (1975) has provided an informative overview of the process from various patents. The salient feature of the processes are as follows.

The corium layer, after being split from the hide, is extensively washed and then mechanically comminuted. The diced collagen is allowed to swell under the controlled conditions of the medium, which comprises an ethyl alcohol-water system and is centrifuged intermittently. The collagen is then treated with HCl at a pH of 1.6-2.6 to allow it to react with the available NH, groups of the collagen. This reaction forms a water-insoluble, ionizable, salt of collagen containing about 0.4-0.7 mmol of acid (HCl/g of collagen). By varying the acid concentration, microcrystalline collagen with different functional properties can be produced. The temperature is maintained below 30°C during this process. The


cake so obtained is air-dried or freeze-dried before milling into a definitive physical form and packing.

Besides HCl, other acids (sulfuric, acetic, and lactic acids) can also be used to produce corresponding partial-acid salts. However, from a practical and commercial viewpoint, HCl is preferred over other acids for most end uses of the resulting product. The physical and microscopic properties of the microcrystalline collagen and its various uses have been described in detail by Battista (1975). A microcrystalline collagen product is commercially produced under the trade name of Avitene.

C. VARIOUS USES OF COLLAGEN AS GELATIN

There are three major areas where hide collagen is used extensively in the form of gelatin: in various food products (Gotthoffer, 1945; Idson and Braswell, 1957; Battista, 1975; Courts, 1977), in photography (Cox, 1972; Rose, 1977), and in pharmaceuticals (Chvapil et al., 1973; Wood, 1977; Bhandari, 1978). According to Idson and Braswell (1957), 65% of the total annual production of gelatin in the United States (- 50 million lb) is consumed in edible products such as desserts, marshmallows, candy, consomme, bakery foods, jellied meat, ice cream, and other dairy products, 20% is used in photography, and 10% in pharmaceutical capsules, ointments, cosmetics, coatings, and other emulsions. Extensive information is available on various food uses of collagen in the form of gelatin (Bennett, 1921; Bogue, 1922; Alexander, 1923; Idson and Braswell, 1957; Stainsby, 1958; Jones, 1977; Howell, 1978; Courts, 1980).

The use of collagen as gelatin has certain inherent nutritional implicatons. For instance, cattle hide trimmings are treated wtih lime solution (containing some other salts) for a prolonged period to facilitate solubilization of collagen. The essential basic amino acids (e.g., lysine, arginine, histidine) are deaminated (Theis and Jacoby, 1941) or decomposed (Highberger and Stecker, 1941) and the essential hydroxy amino acid (threonine) is destroyed (Bowes et al., 1953) during this process. Probably that is why all of these amino acids (except arginine) have been found limiting in gelatin as evaluated by nutritional trials (Chapman et al., 1959; Rama-Rao et al., 1964).

Besides, alkali treatment has been found to induce some other undesirable changes in proteins. These include racemization of amino acids (Masters and Friedman, 1979, 1980; Smith and Silva de Sol, 1980) and formation of lysinoalanine (Sternberg and Kim, 1977; Friedman, 1978). According to Fried- man et al. (1981), hydroxide ion-catalyzed p-elimination reactions of serine, threonine, and cystine produce a dehydroalanine intermediate containing a conjugated carbon-carbon double bond (because of the double bond character of the peptide bond). This intermediate then reacts with the E-NH, group of lysine to form lysinoalanine. The changes induced by alkali in a protein also have nutri-


tional implications. For example, metabolism of the protein and the physiological utilization of amino acids are reduced to a great extent (Woodard et al., 1975; Gould and MacGregor, 1977; Friedman, 1977; Friedman et al., 1981).

In view of these facts, it seems more appropriate to find various food uses of collagen in its native fibrous form. Although Elias et al. (1970) pointed out some possible uses of collagen in fiber or granule form in meat products, little is known about the functional behavior of fibrous collagen in different food systems. On account of its unique biophysical properties, fibrous collagen could be utilized efficiently to function as a water binder, extender, moisturizer, tex- turizer, and emulsifier in different food systems. Studies have recently been initiated to learn how fibrous collagen will function in various food systems.

D. POTENTIAL USES IN FOOD SYSTEMS

Investigations are being carried out at the Oklahoma Agricultural Experiment Station, Stillwater, Oklahoma, on various potential uses of five types of food- grade fibrous collagen (produced by the Eastern Regional Research Laboratory) in various food systems. The main emphasis so far has been on the production of “all-beef‘’ sausages and bakery products, with the substitution of food-grade collagen at different levels (Henrickson, 1980). The major studies made on these aspects are described below.

Schalk (1980) investigated the effect of food-grade collagen substitution on some of the functional properties of coarse-beef bologna by replacing lean meat at 10, 20, and 30% levels. He found no significant difference in the volume change, shrinkage (wrinkling), emulsion stability, or the texture of the final cooked product as compared to control samples (Fig. 21), except that color a-

FIG. 21. Coarse bologna sausages, prepared from beef with 10, 20, and 30% food-grade collagen from hide added. The cross-sectional area and external surface appearance show no difference. Courtesy of Schalk (1980).


FIG. 22. Microscopic view of fine bologna sausage emulsion containing 15% food-grade fibrous collagen from cattle hide (A) and the control (B). There was no apparent difference in emulsion characteristics and the distribution of fat droplets (arrow) in either case (magnification X 320). Courtesy of Gielissen (1981).

values (redness) decreased with increased supplementation of collagen. This study suggested that hide collagen is as good as water binder and fat emulsifier as other proteins of the lean meat such as actomyosin, or that the meat used contained more actomyosin than needed to form the emulsion. In a similar study, Gielissen (198 1) prepared fine-emulsion bologna sausages by incorporating collagen (product No. 1) at 5 , 10, and 15% levels, replacing lean meat, but keeping the fat content constant at 25%. In each case the emulsion was found to be stable, except at higher levels of collagen where the emulsion stability tended to decline slightly. The microscopic examination of various emulsions did not show any adverse effect of collagen on the diameter of fat droplets (Fig. 22).

These findings are at variance with those of Saffle et al. (1964) and Maurer and Baker (1966), who considered collagen detrimental to the emulsifying capacity of poultry meat. The high collagen content (>15%) in sausages has been regarded as the causative factor of gel pockets, wrinkling of the outer skin, poor peelability, and unstable batters when the product is cooked at a temperature above 65°C (Saffle et al., 1964; Kramlich, 1971). On the contrary, Hamm (1972) found the fewest gel pockets in frankfurter-type sausages in the temperature range of 65-85°C. A recent study by Wiley et al. (1979) indicated that the incidence of gel-pocket formation may increase only if the soluble collagen


content is high in the sausage, whereas total collagen or insoluble collagen content bears little relationship with gel-pocket formation.

Slight devaluation of the surface appearance of the cross-sectional area, caused by gel formation, should not be overemphasized in view of certain intrinsic merits of the gel. For example, several studies have shown that protein hydrolysates (Bishov et al., 1967; Bishov and Henick, 1972, 1975) or certain amino acids (Marcuse, 1962; Chang and Linn, 1964; Karel et al., 1966; Hayes et al., 1977) act as synergists for the naturally occurring antioxidants (vitamin E) in retarding the autoxidative changes in the lipid fractions. Karel et al. (1975) have shown that free radicals formed during autoxidation of unsaturated fat are transferred to protein. An electron-spin resonance spectroscopic study by Uchiyama and Uchiyama (1979, 198 1) indicated that free radicals were produced directly and retained in the protein fraction mainly during heating or y irradiation. Free radical production was highest in lysine, followed by tryptophan, phenylalanine, glutamic acid, methionine, and tyrosine. Other amino acids had zero value. Whether or not these trace radicals have any physiological implication seems to be a controversial issue. Although Renner and Reichelt (1973) found no evidence of toxic effect in rats due to feeding diets containing trace radicals, other workers believe that the formation of free radicals in food is the causative factor of mutagenicity (Kosuge et al., 1980; Uyteta et al., 1978; Imoto, 1979).

Recently Kawashima et al. (1979) provided evidence that a collagen hydrolysate (gelatin), especially the fraction with MW 1300-2500, exhibited an outstanding synergistic effect in depressing autoxidation of unsaturated fatty acids in lard. The synergistic effect of the hydrolysate has been assigned to the proline content (Bishov and Henick, 1972), which is extraordinarily high in collagen. Possibly, proline nitroxide formed from proline acts as a synergist in the oxidiz- ing system (Van der Veen et al., 1970; J . S . Lin et al., 1974). Another possibility may be the complex brown pigments, formed by Maillard-type reactions between reducing sugars and amino acids on heating in the presence of organic acids. Despite the biological significance of such reactions (Erbersdobler, 1977) the resulting brown pigments have been found to display a greater antioxidant capacity than some of the synthetic antioxidants (Griffith and Johnson, 1957; Kirigaya et al., 1968; Hwang and Kim, 1973; Itoh et al., 1975; Kawashima et al., 1977; Tufail, 1977; Lingnert, 1980; Eichner, 1980).

Ebro et al. (1979, 1980) conducted a series of studies using food-grade collagen in various bakery products such as beef loaves, whole wheat muffins, sweet wheat loaf (loaf bread), corn meal muffins, plain cakes, applesauce and carrot cake, oatmeal cookies, and plain and whole wheat spatzle (German noo- dles). These studies found little adverse effect of five types of collagen products on certain quality criteria (e.g., juiciness, chewability) of beef loaves, whereas the overall scores for texture and flavor were higher for loaves containing col-


lagen product No. 4. The 20% addition level gave better firmness to the beef loaf than 10 or 30% levels. Objective measurement of loaf volume and color did not reveal any significant influence of the collagen level or product type using the volume index, lightness index, or dominant wavelength of beef loaf.

Ebro et al. (1980) have shown that substitution of air-dried collagen into a plain muffin formulation was not encouraging because the aroma was not acceptable, although the cellular structure was comparable with the reference samples. On the other hand, whole wheat muffins, containing 5% collagen, were as good as the reference samples in aroma but the texture was grainy. This was also true for sweet whole wheat loaf. The organoleptic characteristics of corn meal muffins, containing 10% collagen, were rated equal to the reference samples at the higher levels, but the quality tended to decline. Studies on cakes suggested that white cake is not a suitable medium for collagen supplementation because the granular nature of air-dried collagen resists proper blending with plain cake, which is supposed to have a velvety smooth texture. However, carrot and applesauce cakes containing raisins and nuts appear to be an appropriate medium for fibrous collagen supplementation.

IX. RESEARCH NEEDS

A review by Asghar and Pearson (1980) indicated that the tenderness of meat has been viewed traditionally with reference to the amount or solubility of the connective tissue, yet the opinions of different workers have always been con- flicting. Microscopic differences in collagen of muscle from the same lamb breed (Poll Merino X Dorset Horn) has been reported as having some “fibrillar” collagen characteristics, whereas others had “smooth” appearances (Asghar, 1969; Asghar and F. M. Yeates, 1968, 1979b). But their significance in relation to tenderness is not known. Fortunately, the discovery of genetically different collagen types (such as collagen type I, [~xI(I)]~[a2(1)], type 11, [~x1(11)]~, type 111, [~x1(111)]~, type IV, [cxl(IV)l3, and perhaps others (Eyre, 1980)), which are believed to be produced by different nonallelic structural genes (Harwood, 1979) in different tissues of various species, has opened new avenues by which meat scientists can approach the issue of meat tenderness. First, attempts can now be made to discover if a correlation exists between the type of collagen in a muscle and tenderness. Second, attention may be directed to exploring whether or not all types of collagen have the same potentialities of forming cross-linkages, which become thermally stable with advancing biological age of the animal.

The present state of knowledge regarding the chemical nature of the cross- linkages in collagen has already been discussed in Section II1,D. It may, however, be realized that most of the information on this aspect has been derived from


the studies of collagen from various tissues other than muscle. It is not clearly known what types of cross-linkages predominate in intramuscular collagen from different species of meat animals. A detailed account of the chemistry and biochemistry of the postmortem aging process presented earlier (Asghar and Yeates, 1978) has indicated inconsistent findings of different researchers who relied on the solubility criterion of connective tissue. Since the chemistry of various cross-linkages in collagen is now well established (Tanzer, 1976), it would be worthwhile to examine the postmortem changes in connective tissue of muscle in terms of the chemical linkages which are labile to the actions of lactic acid and/or lysosomal cathepsins.

The studies on type I collagen mRNAs have successfully established the gene coding for pro-al(I) and pro-a2(I) chains. This may help in constructing a structural map of monocistronic procollagen mRNA and its probable translation products (Hanvood, 1979). Similar approaches may be applied to investigate mRNAs for other types of collagen for better understanding of their transcrip- tional and translational control at subcellular levels. Once the fundamental information on the factors responsible for gene coding on individual types of collagen is elucidated, the appropriate genes may be activated to encourage specific types of collagen (Harwood, 1979). Since the synthesis of different types of collagen is under genetic control, it seems feasible to screen the breeding stock for a specific type of collagen which may be associated with tenderness. Thus, by proper genetic manipulation, meat animal breeds with desired collagen types may be evolved.

Another important area which merits further investigation is the functional properties of fibrous collagen in different food systems. It has already been mentioned (Section VI,A) that one of the most important functional properties of fibrous collagen in a food system is the water-holding capacity. However, little is known about the different variables affecting this property of collagen except the pH and a few ions. It is also not clear as to how different chemically modified collagen derivatives (e.g., ether, ester, phosphate, sulfate) would behave as moisturizers, binders, emulsifiers, texturizers, and extenders in different food systems. For this type of investigation, the experimental methodology available on starch (Kerr, 1950; Roberts, 1967; Hamilton and Paschall, 1967; Kruger and Rutenberg, 1967; Knight, 1967; Radley, 1968), casein (Southward and Gold- man, 1975, 1978), soy proteins (Meyer and Williams, 1977; Richardson, 1977), and cellulose (Ott et al., 1954; Honeyman, 1959; Yarsley et al. , 1964) derivatives may well serve as models for the development and evaluation of fibrous collagen derivatives. The modifications can possibly be achieved with different degrees of substitution at hydroxyl groups of the carbohydrate moiety, associated with collagen molecules to impart desired functional properties needed for different food systems.


ACKNOWLEDGMENTS

Journal Services Paper 896 of the Oklahoma Agricultural Experiment Station. Financed in part by the USDA Science and Education Administration, Eastem Regional Research Center, Philadelphia, Coop. Agreement 58-32-U-4-8-2. The authors wish to acknowledge the editorial assistance of Dr. George Gorin, Deborah Doray, Susan Johnson, Dorothy Sipe, and Lyn Sweet.

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