THE JOURNAI, OF ~~I~LOGICAL CHEMISTRY Vol. 241, No. 2, Issue of January 25, 1966

Printed in U.S.A.

Amino Acid Sequence of Chicken Heart Cytochrome c

(Received for publication, August 30, 1965)

s. K. CHART” ASD E. RIARGOLIASH

From the Biochemical Research Department, Abbott Laboratories, North Chicago, Illinois 60064

SUMMARY

The complete amino acid sequence of chicken heart cyto- chrome c has been established. This primary structure is typically that of a “mammalian-type” cytochrome c showing the characteristic groupings of hydrophobic and basic res- idues, and, like the other cytochromes c from vertebrate species, has an acetylated amino-terminal residue. Chicken heart cytochrome c differs from the horse, beef, human, pig, tuna, moth, and bakers’ yeast iso-1-cytochrome c proteins by 11, 9, 12, 9, 19, 28, and 45 residues, respectively.

Extensive comparisons of primary structures, as well as of physicochemical and enzymic properties, within a set of homolo- gous proteins, can be expected to yield information on the degree of variation compatible with the same function, indicate those areas and those properties which are constantly required for function, and make possible studies of the relations between evolutionary changes in protein structure and the evolution of species. Cytochrome c has proved to be particularly amenable to such an approach (see Margoliash and Schejter (l)), and the amino acid sequences of the proteins from horse (2), man (3), beef (4), pig (5), tuna (6, 7), bakers’ yeast (8), and a moth, Sumia cynthia (9), have already been reported. Thus, among vertebrate cytochromes c, only mammalian and a single fish protein have so far been studied. Since cytochromes c from the other three vertebrate classes represent a necessary extension of the phylogenetic data and are likely to show different residue variations in different positions than those previously observed, progress in this area requires knowledge of representative avian, reptilian, and amphibian proteins.

The present paper is an initial report of a study of bird cyto- chromes c and presents the complete structure of the chicken protein. The amino acid sequence in the region of heme attach- ment for this protein has been determined by Tuppy and Pale& (10) as

Val-Gln-Lys-Cys-Ser-Gln-CysHis-Thr-Val-Glu

I-heme--/

EXPERIMENTAL PROCEDURE

Chicken cytochrome c was prepared and crystallized from fresh frozen material (II), redissolved, thoroughly dialyzed, and

* Present address, Department of Biochemistry, University of Kentucky Medical School, Lexington, Kentucky.

lyophilized. The total amino acid composition, iron content, and dry weight were determined as previously described (9). A sample of 100 pmoles of cytochrome c was digested at 38” with 78 mg of three times crystallized a-chymotrypsin, which was added successively in three equal portions for a total of 30 hours. Similarly, 15 pmoles of cytochrome c were digested with t.rypsin (twice crystallized) which had been treated to minimize the contaminating chymotryptic activity according to Redfield and Anfinsen (12). Trypsin was added to the digestion mixt,ure in three equal portions, and digestion was allowed to proceed for 9 hours at 38”. The enzymes were purchased from the Worthing- ton Biochemical Corporation. The protein was subjected to Edman degradation; identification of the phenylthiohydantoin derivatives was attempted by paper chromatography (13, 14).

Chymotryptic and tryptic digests of cytochrome c were frac- tionated by ion exchange column chromatography as given in Figs. 1 and 2, with t,he use of Dowex 50-X8 for the chymotryptic digest and nowex 50-X2 for the tryptic digest. Further peptide purifications were carried out by gel filtration on Sephadex G-25, by paper electrophoresis and chromatography, or by combina- tions of these techniques (9). The purity of fractions was moni- tored by paper electrophoresis-chromatography, with t,he use of the various color reactions previously employed (9). Nin- hydrin-negative peptides were detected by the starch-iodine reaction (15). Hydrazinolysis of peptides was performed accord- ing to Funatsu, Tsugita, and Fraenkal-Conrat (16). Sequential degradations of peptides were carried out by the Edman proce- dure as modified by Hirs, Moore, and Stein (17) and Konigs- berg and Hill (18). Amino acid compositions of peptides were determined on acid or total enzymic digests (leucine aminopepti- dase) in the Beckman/Spinco automatic amino acid analyzer. Yields were calculated from the analyses of the purified peptides, and represent the minimal recovery. Digestions of peptides with trypsin, carboxypeptidase A, leucine aminopeptidase, elastase, and papain (Worthington) were performed as previously described (9). The amino acids released by leucine amino- peptidase and carboxypeptidase A were determined by amino acid analysis.

One peptide (3.0 pmoles) was subjected to partial hydrolysis in 10 ml of 0.1 M acetic acid, under reduced pressure (5 mm of Hg) for 6 hours at 105” (see Table II).

RESULTS

Molecular Weight, Amino Acid Composition, and Edman Degradation of Chicken Heart Cytochrome c

The molecular weight calculated from the iron content of 0.45% (12,411), assuming one heme per molecule of protein, is in

507

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508 Chicken Heart Cytochrome c Vol. 241, No. 2

good agreement with that calculated from the amino acid se- quence (12,222) (see Fig. 3). The amino acid composition of the protein, determined directly on acid hydrolysates, is reported in Table I and is identical with that obtained from the amino acid sequence. A single step of Edman degradation on the intact protein failed to reveal any phenthiohydantoin derivative.

Separation and Purijcation of Peptides from Chymotryptic and

Tryptic Digests

Nomenclature--The elution patterns of peptides obtained by column chromatography of the chymotryptic and tryptic digests are given in Figs. 1 and 2, respectively. It may be noted that the tryptic heme peptide was recovered in good yield from chromatographic Peak XIII (Fig. 2 and Table XII), while the chymotryptic heme peptide remained adsorbed to the top of the resin column throughout the chromatography. The peptides were further purified, when necessary, as indicated in Tables II to XII, which summarize the data used in establishing the amino acid sequences. The purification procedures used in each case are indicated by the symbols PC for paper chromatography, PE for paper electrophoresis, and S for Sephadex column chroma- tography. The tables also list the chromatographic (ch) and electrophoretic (el) mobilities of the peptides on paper in centi-

TABLE I

Analysis of acid hydrolysates* of chicken heart cytochrome c

Amino acid unino acid residues

Lysine Histidine Arginine Aspartic acid. Threonine Serine Glutamic acid. Proline Glycine. Alanine........... Half-cystine Valine Methionine Isoleucine Leucine Tyrosine. Phenylalanine Tryptophant.. Hemet............ Hydroxyl t Acetylt Total

g/l00 g protein

19.44 3.37 2.41 8.00 6.71 2.76

10.14 2.43 6.16 2.89 1.68 2.40 2.29 6.15 5.37 5.34 4.82 1.38 5.09 0.14 0.22

99.19 -

From analysis F ‘mm amino acid sequence

Amino acid residues per molecule of protein

18.6 3.0 1.9 8.6 8.2 3.9 9.7 3.1

13.3 5.0

3.0 2.1 6.7 5.9 4.0 4.0

18 3 2 9 8 4

10 3

13 5 2 3 2 7 6 4 4 1

104

* Samples of the intact protein were hydrolyzed under reduced pressure in three times glass-distilled 6 N HCI for 20, 40, and 72 hours. Duplicate analyses were performed on each hydrolysate. The amount of the sample represented in each aliquot was calcu- lated from the dry weight of the sample. The data reported are derived from the average or the extrapolated value of all the analyses.

t Calculated, assuming the appropriate number of residues per molecule.

r

EFFLUENT VOLUME IN LITERS

FIG. 1. Elution pattern of peptides from a chymotryptic digest of chicken heart cytochrome c. The digest (100 Nmoles) was chromatographed on a column of Dowex 50-X8 (3.7 X 150 cm) with a linear gradient established between pyridine-acetic buffer at pII 3.1 (0.2 M) and pH 4.8 (2.0 M). The column was operated at 40”. The line across the top of the pattern indicates the pH of the effluent fractions. The thick lines on the abscissa mark the fractions pooled and numbered in Roman numerals.

5.0

s

2

b I *

3.0

EFFLUENT VOLUME IN LITERS

FIG. 2. Elution pattern of peptides from a tryptic digest of chicken heart cytochrome c. The digest (15 pmoles) was chroma- tographed on a column of Dowex 50-X2 (G.9 X 150 cm) under the conditions given for the chymotryptic digest in Fig. 1. The symbols are those used in Fig. 1.

meters under the following standard conditions: electrophoresis, pH 6.4, pyridine-acetic acid buffer (19), 19 volts per cm, 90 min; chromatography, l-butanol-acetic acid-water solvent (20), 16 hours. A zero (0) indicates no electrophoretic movement; a

minus sign ( -), movement toward the cathode; and a plus sign (+), movement toward the anode. In Tables II to XI, the purification procedures, yield, electrophoretic-chromatographic

mobilities, and ninhydrin color, as well as other various reactions with specific amino acid residues, are listed inside brackets, in

this order, immediately following the amino acid composition of the peptide. The peptides are denoted by Roman numerals according to the column chromatographic fractions in which they

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Issue of January 25, 1966 S. K. Chan and E. Margoliash 509

emerged, T referring to peptides from the tryptic digest and C to those from the chymotryptic digest. When more than one peptide was recovered in a single chromatographic fraction, they are distinguished by lower case letters. Fragments derived from the initial chymotryptic peptides by digestion with trypsin, elastase, dilute acetic acid, and papain are denoted by T, E, A, and P, respectively. For the digestion of peptides with carboxy- peptidase A and leucine aminopeptidase, the values reported in the tables are ratios of the molar amounts of amino acids re- leased by the exopeptidases. These ratios were calculated by taking the quantity for the residue released in the largest amount as unity. In all tables and in the text, the numbers given in parentheses are the assumed stoichiometric numbers of residues per molecule of -pure peptide. In reporting Edman degrada- tions, the residues and the numbers marked in boldface type correspond to the residue removed at each step.

Amino Acid Sequences of Peptides from Chymotryptic Digest

The amino acid sequence of chicken heart cytochrome c is given in Fig. 3, and the individual peptides are discussed in the order in which they appear in the over-all sequence, starting from the amino-terminal end.

Residues 1 to 10: AcetylGly-Asp-Ile-Glu-Lys-Gly-Lys-Lys- Ile-Phe (Table II, Peptide C-Fa)-Peptide C-Va was neutral, although the amino acid composition shows an excess of basic over acidic residues. This, together with the failure to obtain any free amino acids on digestion with leucine aminopeptidase and any change in composition following Edman degradation of either Peptide C-Va or tryptic fragment T-l, indicates a blocked amino-terminal residue. Three fragments isolated from the partial acid digest of T-l, A-3, A-4, and A-2 were ninhydrin- negative. The results obtained by hydrazinolysis of A-3 and the compositions of A-3 and A-4 established the sequence acetyl- Gly-Asp .

The composition of A-l, its electrophoretic neutrality, and one step of Edman degradation, together with the composition of A-2, yielded the sequence of the 3 other residues of the original

tryptic fragment T-l. Carboxypeptidase A digestion of Pep- tide C-Va liberated isoleucine and phenylalanine, the 2 residues comprising the composition of tryptic fragment T-2, which from chymotryptic specificity must have the structure Ile-Phe and represents the carboxyl-terminal sequence of the original peptide. The remaining tryptic fragments T-3 and T-4 must therefore derive from the center of Peptide C-Va. Their compositions and one step of Edman degradation on T-3 establish the sequence Gly-Lys-Lys.

AcetylGly-Asp-Ile-G1u-Lys-Gly-Lys-Lys-Ile-Phe-Val-G1n- 10

His-Lys-Thr-Gly-Pro-Asn-Leu-His-Gly-Leu-Phe-Gly-~ rg- 30

Lys-Thr-Gly-Gln-Ala-Glu-Gly-Phe-Ser-Tyr-Thr-Asp-Ala- 40 50

Asn-Lys-Asn-Lys-Gly-Ile-Thr-Trp-Gly-Glu-Asp-Thr-Leu- 60

Met- Glu-Tyr-Leu-Glu-Asn-Pro-Lys-Lys-Tyr-Ile-Pro-Gly- 70

Thr-Lys-Met-Ile-Phe-Ala-Gly-Ile-Lys-Lys-Lys-Ser-Glu- 80 90

Arg-Val-Asp-Leu-Ile-Ala-Tyr-Leu-Lys-Asp-Ala-Thr-Ser- 100

LysCOOH 104

FIG. 3. Amino acid sequence of chicken heart cytochrome c. The hydrophobic residues leucine, isoleucine, valine, methionine, tyrosine, phenylalanine, and tryptophan are shown in boldface type; the basic residues lysine, arginine, and histidine are shown in italics.

TABLE II

Amino acid sequence of residues 1 to 10

Sequence: AcetylGly-Asp-Ile-Glu-Lys-Gly-Lys-Lys-Ile-Phe

Peptide

C-Va

Leucine aminopeptidase Carboxypeptidase A Edman degradation

T-l

A-l Edman I

A-2 Edman degradation

A-3 Hydrazinolysis

A-4 T-2 T-3

Edman I T-4

Gly, 1.65(2); Asp, 1.01(l); Ile, 2.12(2); Glu, 1.11(l); Lys, 3.00(3); Phe, 1.10(l) [PE, PC; 20%; el, 0; ch, 12.5; blue]

No free amino acids released Ile, 0.81; Phe, 1.00 No change in amino acid composition Gly, 1.21(l); Asp, 0.77(l); Ile, 1.08(l); Glu, 1.16(l); Lys, 1.07(l) [PE, PC; el, +7.5; ch, 17.9; ninhy-

drin-negative] Ile, 0.98(l); Glu, 0.98(l); Lys, 1.04(l) [PC; el, 0; ch, 10.5; blue] Ile, 0.06; Glu, 1.00(l); Lys, not determined Gly, 1.13(l); Asp, 1.06(l); Ile, 0.92(l); Glu, 0.90(l) [PC; el, f3.3; ch, 18.0; ninhydrin-negative] No change in amino acid composition Gly, 1.00(l); Asp, 1.00(l) [PC; el, +4.5; ch, 30.0; ninhydrin-negative] Acetylhydrazide, glycylhydrazide, and free aspartic acid Gly [PC; el, +3.0; ch, 33.0; ninhydrin-negative] Ile, 0.88(l); Phe, 1.11(l) [PE, PC; el, 0; ch, 36.3; blue] Gly, 1.00(l); Lys, 1.91(a) [PE, el, -14.5; ch, 1.0; yellow] Gly, 0.20; Lys, 2.00(2) Gly, 0.96(l); Lys, 1.04(l) [PE; el, -10.0; ch, 3.5; yellow]

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510 Chicken Heart Cytochrome c Vol. 241, No. 2

Residues 11 to g%?-This area is contained in the heme peptide which was not recovered from the chymotryptic digest. How- ever, since the amino acid sequence of this segment from residues 11 to 21 has been previously determined by Tuppy and Pale& (10) and is covered by two tryptic peptides, T-Xb and T-XIIIa (see Table XII), no further work was undertaken. It may be noted that the composition of Peptides T-Xb and T-XIIIa to- gether with the results obtained by Tuppy and Pale& (10) identify residue 22 as lysine.

Residues 23 to 26: Gly-Gly-Lys-His (Peptide C-XIII)-Pep- tide C-XIII (Gly, 1.87(2); Lys, 0.96(l); His, 1.16(l); el, -14.0; ch, 2.0) gave a yellow ninhydrin color on paper. It was purified by paper electrophoresis in a yield of 4%. Tryptic digestion gave free histidine, indicating that this residue was carboxyl- terminal. The one other tryptic fragment was basic (el, -6.5; ch, 7.0) and also gave a yellow ninhydrin color. Two steps of Edman degradation on Peptide C-XIII established the over-all sequence.

Step I: Gly, 0.93(l); Lys, 1.09(l); His, 1.15(l). Step II: Gly, 0.26; Lys, 1.00(l); His, 1.00(l). Residues 27 to SS: Lys-Thr-Gly-Pro-Asn-Leu-His (Table III,

Peptides C-IXb and C-VI)-Carboxypeptidase A digestion of Peptide C-IXb gave leucine and histidine in equal quantities, while extensive hydrolysis with chymotrypsin yielded free histi- dine and a basic peptide. The carboxyl-terminal sequence of this

peptide is therefore Leu-His. Three steps of Edman degrada- tion yielded the sequence Lys-Thr-Gly, and since digestion with leucine aminopeptidase liberated only lysine and threonine, proline must be the 4th residue from the amino-terminal end (21). The aspartyl residue must then follow the proline and precede the Leu-His sequence. Peptide C-VI has the same composition as Peptide C-IXb except that it lacks leucine and histidine. Both Peptide C-VI and the peptide obtained by chymotryptic digestion of Peptide C-IXb are basic, showing the aspartyl residue to be present as the amide.

Residues Sk to 36: Gly-Leu-Phe (Peptide C-II)-Peptide C-II (Gly, 1.16(l); Leu, 0.79(l); Phe, 1.06(l); el, 0; ch, 34.0) gave a yellow ninhydrin color. It was purified by paper chroma- tography and obtained in 15% yield. Two steps of Edman degradation established its sequence, as follows.

Step I: Gly, 0.37; Leu, 1.05(l); Phe, 0.94(l). Step II: Gly, 0.10; Leu, 0.20; Phe, 1.00(l). Residues 37 to 46: Gly-Arg-Lys-Thr-Gly-Gln-Ala-Glu-Gly-

Phe (Table IV, Peptide C-XIc)-The amino- and carboxyl- terminal residues are glycine and phenylalanine, respectively, as shown by Edman degradation and carboxypeptidase A diges- tion. This, and the compositions of tryptic fragments T-l and T-2, indicate the amino-terminal sequence to be Gly-Arg-Lys. Edman degradations of the third tryptic peptide, T-3, together

TABLE III

Amino acid sequence of residues 27 to SS

Sequence : Lys-Thr-Gly-Pro-Asn-Leu-His

Peptide I

C-IXb

Carboxypeptidase A Chymotrypsin Leucine aminopeptidase Edman I Edman II Edman III

C-J’1

Lys, 0.95(l); Thr, 1.02(l); Gly, 0.96(l); Pro, 1.00(l); Asp, 1.16(l); Leu, 0.89(l); His, 1.01(l) [PE, PC; 13%; el, -7.0; ch, 3.0; blue, Pauly]

Leu, 0.90; His, 1.00 His and a basic peptide identified by paper electrophoresis-chromatography Lys, 1.00; Thr, 0.51 Lys, 0.03; Thr, 0.95(l); Gly, 0.97(l); Pro, 0.66(l); Asp, 1.16(l); Leu, 1.11(l); His, 1.15(l) Lys, O.O5;Thr, 0.14; Gly, 1.00(l); Pro, 1.03(l); Asp, 0.89(l); Leu, 1.00(l); His, 1.17(l) Thr, 0.08; Gly, 0.05; Pro, 1.15(l); Asp, 1.04(l) ; Leu, 0.82(l) ; Lys and His not determined Lys, 1.04(l); Thr, 0.87(l); Gly, 1.16(l); Pro, 0.91(l); Asp, 0.99(l); His, 0.03; Ser, 0.08; Glu, 0.25; Ala,

0.08; Leu, 0.07 [PE; 3%; el, -6.0; ch, 5.0; blue] -

TABLE IV

Amino acid sequence of residues S7 to 46 Sequence : Gly-Arg-Lys-Thr-Gly-Gln-Ala-Glu-Gly-Phe

Peptide

C-XC

Carboxypeptidase A Edman I

T-l T-2 T-3

Edman I Edman II Edman III

E-l E-2

Edman I ES3

Gly, 3.06(3); Arg, 1.06(l); Lys, 1.35(l); Thr, 0.95(l); Glu, 2.16(2); Ala, 0.95(l); Phe, 0.91(l) [PC; 16%; el, -5.0; ch, 7.3 yellow, Sakaguchi]

Gly, 0.20; Phe, 1.00 Gly, 2.06(2); Arg, 0.90(l); Lys, 1.00(l); Thr, 1.17(l); Glu, 1.92(2); Ala, 0.97(l); Phe, 0.83(l) Gly, 0.87(l); Arg, 1.13(l) [PE; el, -9.0; ch, 5.2; yellow, Sakaguchi] Lys [PE; el, -13.0; ch, 4.0; blue] Thr, 0.53(l); Gly, 2.14(2); Glu, 1.87(2); Ala, 1.08(l); Phe, 0.78(l) [PE; el, -3.2; ch, 14.4; yellow] Thr, 0.17; Gly, 1.92(2); Glu, 2.00(2); Ala, 1.01(l); Phe, 1.01(l) Thr, 0.11; Gly, 1.20(l); Glu, 1.95(2); Ala, 1.01(l); Phe, 0.84(l) Thr, 0.09; Gly, 1.22(l); Glu, 1.38(l); Ala, 0.99(l); Phe, 0.91(l) Thr, 0.89(l); Gly, 1.18(l); Glu, 1.11(l); Ala, 0.83(l) [PE, PC; el, 0; ch, 11.0; yellow] Glu, 0.95(l); Gly, 1.05(l) [PE, PC; el, +3.0; ch, 8.0; blue] Glu, 0.35; Gly, 1.00(l) Phe [PE, PC; el, 0; ch, 26.0; blue]

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Issue of January 25, 1966 X. K. Chan and E. Margoliash 511

TABLE V

Amino acid sequence of residues 49 to 64

Sequence: Thr-Asp-Ala-Asn-Lys-Asn

Peptide

C-III

Edman I Edman II Edman III

T-l T-2 P-l P-2 P-3

Thr, 1.00(l); Asp, 2.76(3); Ala, 1.13(l); Lys, 1.12(l) [PC; 26% el, 0; ch, 2.0; yellow] .Thr, 0.07; Asp, 3.10(3); Ala, 0.81(l); Lys, 1.09(l) Thr, <O.lO; Asp, 2.10(2); Ala, 0.91(l); Lys not determined Thr, <O.lO; Ala, 0.36; Asp, 1.98(2); Lys not determined Thr, 0.68(l); Asp, 2.10(2); Ala, 0.78(l); Lys, 1.17(l) [PC; el, 0; ch, 4.5; yellow] Asn, identified by paper electrophoresis-chromatography [PC; el, 0; ch, 9.0; orange] Thr; Asp; Ala, identified by paper electrophoresis-chromatography [PE; el, +5.0; ch, 5.2; yellow] Asn; Lys, identified by paper electrophoresis-chromatography (PE; el, -4.0; ch, 4.8; blue] Asn; identified by paper electrophoresis-chromatography [PE; el, 0; ch, 2.0; orange]

Peptide

C-XIIb

Leucine aminopeptidase Edman I Edman II Edman III

i

TABLE VI

Amino acid sequence of residues 56 to 59

Sequence : Lys-Gly-Ile-Thr-Trp

Lys, 1.14(l); Thr, 0.83(l); Gly, 1.03(l); Ile, 1.00(l); Ser, 0.01; Leu, 0.03 [PC; 25%; el, -4.5; ch, 18.0; blue, Ehrlich]

Lys, 1.05(l); Thr, 1.00(l); Gly, 1.04(l); Ile, 1.11(l); Trp, 0.79(l) Lys, 0.01; Gly, 1.07(l); Ile, 1.07(l); Thr, 0.88(l) Gly, 0.49; Ile, 1.00(l); Thr, 0.73(l); Lys not determined Gly, 0.20; He, 0.19; Thr, 1.00(l); Lys not determined

TABLE VII

Amino acid sequence of residues 60 to 65

Sequence: Gly-Glu-Asp-Thr-Leu-Met

Peptide

C-l

Carboxypeptidase A Chymotrypsin

Edman I Edman II Edman III

Gly, 1.07(l); Glu, 1.07(l); Asp, 1.09(l); Thr, 0.99(l); Leu, 0.95(l); Met, 0.88(l); Lys, 0.11; Ser, 0.11; Ala, 0.12; Ile, 0.11 [PE; 10%; el, +8.0; ch, 26.0; yellow, Toennies]

Leu, 0.95; Met, 1.00 Met and an acidic peptide identified by paper electrophoresis-chromatography, which after acid

hydrolysis contained Gly, 0.92(l) ; Glu? 1,12(l) ; Asp, 1.04(l) ; Thr, 0.95(l) ; Leu, 0.97(l) Gly, 0.27; Asp, 0.88(l); Glu, 1.33(l); Thr, 0.84(l); Leu, 1.26(l); Met, 0.68(l) Gly, 0.01; Asp, 0.50; Glu, 1.00(l) ; Thr, 1.01(l) ; Leu, 1.05(l) ; Met, 0.98(l) Gly, 0.12; Asp, 0.57; Glu, 0.45; Thr, 1.04(l); Leu, 0.98(l); Met, 0.86(l)

TABLE VIII

Amino acid sequence of residues 68 to Yk

Sequence : Leu-Glu-Asn-Pro-Lys-Lys-Tyr

Peptide

C-Xb

Leucine aminopeptidase C-XIa

Leucine aminopeptidase T-l

Edman I Edman II

T-2

Leu, 0.90(l); Glu, 0.97(l); Asp, 0.90(l); Pro, 1.39(l); Lys, 1.90(2); Tyr, 0.96(l) [PE, PC; 13%; el, -4.5; ch, 7.5; blue, Pauly]

Leu, 1.00; Glu, 0.60 Glu, 1.23(l); Asp, 1.04(l); Pro, 1.01(l); Lys, 1.71(2); Tyr, 0.99(l); His, 0.05; Arg, 0.08; Thr, 0.07; Ser,

0.04; Gly, 0.13; Ala, 0.04; Val, 0.05 [PC; 13%; el, -5.2; ch, 5.0; blue, Pauly] Glu, identified by paper electrophoresis-chromatography Glu, 1.10(l); Asp, 0.92(l); Pro, 0.99(l); Lys, 1.00(l) [PE; el, -8.3; ch, 8.0; blue] Glu, 0.53; Asp, 1.24(l); Pro, 1.11(l); Lys, 1.00(l) Glu, 0.20; Asp, 0.40; Pro, 1.00(l); Lys, 1.00(l) Lys, 1.00(l); Tyr, 1.00(l) [PE; el, -8.3; ch, 8.0; blue]

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512 Chicken Heart Cytochrome c Vol. 241, No. 2

with the composition and electrophoretic neutrality of fragment drin coloration and was purified in 50% yield. One step of E-l, obtained by elastase digestion of T-l, showed the amino- Edman degradation established the sequence, as follows. terminal sequence of T-l to be Thr-Gly-Gln-Ala. The other Step I: Ser, 0.10; Tyr, 1.00(l). two products of elastase digestion of T-3 were the dipeptide, Residues 49 to 54: Thr-Asp-Ala-Asn-Lys-Asn (Table V,

Glu-Gly (E-2, sequence by Edman degradation), and free phen- Peptide C-III)-Peptide C-III is neutral, and 2 of the 3 aspartyl ylalanine (E-3). This establishes the over-all sequence given, residues must therefore be amidated. Since the amino-terminal since phenylalanine must be carboxyl-terminal in T-l as well as sequence is Thr-Asp-Ala as determined by Edman degradations, in C-XIc. and since tryptic digestion of C-III yielded free asparagine and a

Residues 47 to 48: Ser-Tyr (Peptide C-VIIb)-This dipeptide neutral peptide, the carboxyl-terminal sequence must be Lys- (Ser, 0.77(l) ; Tyr, 1.22(l) ; el, 0; ch, 21.3) gave a yellow ninhy- Asn. The over-all structure is thus established except for the

TABLE IX

Amino acid sequence of residues 7.5 to 80

Sequence : Ile-Pro-Gly-Thr-Lys-Met

Peptide

C-VC

Tryptic digestion Edman I Edman II Edman III

Ile, 0.94(l); Pro, 1.00(l); Gly, 0.92(l); Thr, 1.06(l); Lys, 1.10(l); Met, 0.92(l); Asp, 0.06; Ser, 0.01 [PE, PC; 25%; el, -5.0; ch, 15.5; blue, Toennies]

Met and a basic peptide, identified by paper electrophoresis-chromatography Ile, 0.10; Pro, 1.26(l); Gly, 082(l); Thr, 1.00(l); Met, 0.72(l); Lys not determined Ile, 0.10; Pro, 0.22; Gly, 1.02(l); Thr, 1.09(l); Met, O.%(l); Lys not determined Ile, 0.10; Pro, 0.10; Gly, 0.52; Thr, 0.98(l); Met, 1.01(l); Lys not determined

Peptide

C-Xa

Carboxypeptidase A (20 hrs)

C-VIIa

Carboxypeptidase A (20 hrs)

T-l Edman I

T-2 Edman I

-

TABLE X

Amino acid sequence of residues 88 to 94

Sequence : Lys-Ser-Glu-Arg-Val-Asp-Leu

Lys, 1.32(l); Ser, 0.83(l); Glu, 1.22(l); Arg, 0.87(l); Val, 0.91(l); Asp, 0.95(l); Leu, 0.92(l); Thr, 0.01; Gly, 0.04; Ala, 0.01 [PE, PC; 4%; el, 0; ch, 7.5; blue, Sakaguchi]

Asp, 0.15; Leu, 1.00

Ser, 0.80(l); Glu, 1.07(l); Arg, 1.06(l); Val, 1.37(l); Asp, 1.12(l); Leu, 1.17(l); Lys, 0.05; Gly, 0.04; Iie, 0.02 [PC; 22%; el, -3.0; ch, 16.8; yellow, Sakaguchi]

Asp, 0.14; Leu, 1.00

Ser, 0.73(l); Glu, 0.82(l); Arg, 1.18(l) [PC; el, 0; ch, 7.5; yellow, Sakaguchi] Set-, 0.10; Glu, 0.92(l); Arg, 1.10(l) Val, 1.08(l); Asp, 1.00(l); Leu, 0.93(l) [PC; el, +8.0; el, 29.8; blue] Val, 0.10; Asp, 0.97(l); Leu, 1.03(l)

TABLE XI

Amino acid sequence of residues 98 to 104

Sequence : Leu-Lys-Asp-Ala-Thr-Ser-Lys

Peptide

C-VIII Leu, 1.02(l); Lys, 2.19(2); Asp, 0.93(l); Ala, 1.03(l); Thr, 0.94(l); Ser, 0.89(l); [PC; 16%; el, -5.0; ch, 5.0; blue]

Edman I Leu, 0.10; Lys, 2.00(2); Asp, 1.10(l); Ala, 1.10(l); Thr, 0.90(l); Ser, 0.90(l) T-l Leu, 1.05(l); Lys, 0.95(l) [PC; el, -9.0; ch, 13.5; blue] T-2 Asp, 1.04(l); Ala, 1.12(l); Thr, 0.96(l); Ser, 0.94(l); Lys not determined [PC; el, 0; ch, 5.0; blue]

Hydrazinolysis Free lysine, 0.85* Edman I Asp, 0.12; Ala, 1.09(l); Thr, 0.99(l); Ser, 0.92(l); Lys not determined Edman II Asp, 0.10; Ala, 0.08; Thr, 1.02(l); Ser, 0.99(l); Lys not determined Edman III Asp, 0.10; Ala, 0.10; Thr, 0.10; Ser, 1.00(l); Lys not determined

C-IXa Lys, 2.20(2); Asp, 0.92(l); Ala, 1.01(l); Thr, 1.04(l); Ser, 0.84(l) [PE; PC; 5%; el, -4.7; ch, 2.8; blue] Edman I Lys, 0.92(l); Asp, 1.06(l); Ala, 1.02(l); Thr, 1.02(l); Ser, 0.48(l) Edman II Lys, 1.06(l); Asp, 0.50(l); Ala, 1.02(l); Thr, 0.98(l); Ser, 1.07(l)

* This value is the molar ratio of lysine recovered to the amount of peptide subjected to hydrazinolysis.

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Issue of January 25, 1966 S. K. Chan and E’. Margoliash 513

TABLE XII

Amino acid compositions of tryptic peptides from chicken heart cytochrome c

Peptide Amino acid - - -

T-I T-II T-III T-IV T-V T-VI T-VIIa T-VIIb T-VII& T-VIIIb --

Tryptophan ........... Lysine ............... Histidine .............. A rgmme .............. Aspartic acid. Threonine ............ Swine ................ Glutamic acid, ....... Proline. ............. Glycine Alanine ............... Half-cystine .......... Valine .............. Methionine .......... Is&wine. ............ Leucine .............. Tyrosine .............. Phenylalsnine ......... No. of residues ........ Yield (‘$&). ............ ‘32 (cm). ............... ch (cm). ............... Ninhydrin color ....... Purification procedure

1.11(l) 0.03 0.94(l) 0.94(l) 1.00(l)* (1)

1.24(l) 1.08(l) 1.04(1

0.90(l) 0.09

1.00(l)

1.00(l)

1.11(l) 0.06 0.06 0.06

1.07(l) 0.99(l) 1.02(l)

2.15(2) 1.91(2) 0.85(l) 2.16(2)

0.09 1.99(2) 1.77(2)

1.24(l)

0.91(l) 0.91(l)

0.92(l)

0.12

0.06 0.73(l)

1.11(l)

1.08(l) 1.08(l) 0.92(l)

6

Ji.0 37.2

Blue

0.08

2.85(3) 1.00(l) 1.86(2)

0.94(l) 2.08(Z)

0.99(l) 2.32(2) 2.20(Z)

0.98(l) 0.05 0.09

0.94(l) 1.00(l) 2.32(2) 0.98(l)

1.99(2)

5

J.5 17.9

Blue

5 84

0 5.0 Blue

0.49(l) 0.95(l)

14 28

+2.5 5.0

Yellow S

1 20

-14.2 7.0 Blue

17

$1 21.5 Yellow S

1.01(l)

1.00(l)

1.00(l) 1.09(l)

0.91(l)

5 13

-1.0 21.0 Yellow s, PC

0.91(l)

2.10(2)

3 59

-7.5 4.7

Yellow S

0.85(l) 9

19.8 -1.0

22.3 Yellow s

2 2 3 40 21.9 43

-3.8 -6.6 0 4.8 4.8 6.8 Blue Yellow Yellow PE PE PC

- - Peptide

Amino acid - - T-IXb T-Xa T-Xb T-Xc T-XIa T-XIb T-XIIa T-XIIb T-XIIc T-XIIIa T-XIIIb T-XIV

-- _- Tryptophan ......... Lysine ............... Histidine .............. Arginine .............. Aspartic acid ....... Threonine ............. Swine ................. Glutamic acid., ...... Praline ............... Glycine ............... Alanine ............... Half-cystine Valine ................ Methionine ............ Isoleucine. ........... Leucine .............. Tyrosine .............. Phenylalanine. ........ No. of residues ........ Yield (‘%). ........... ez (cm) ................ ch (cm) ................ Ninhydrin color. ..... Purification procedure

1.15(l) 2.39(2) 1.00(l) 1.87(2) 1.00(l)

1.00(l)

0.90(l) 2.18(2) 2.04(2) 1.00(l) 1.98(2) 1.96(2) 2.04(a)

1.10(l) 0.04 0.90(l) 0.04 0.03 0.89(l) 1.10(l)

1.16(l) 0.84(l)

0.05 0.01 0.02

1.89(2) 1.15(l) 1.19(l)

1.26(l)

0.90,(l) 0.97(l) 1.96(2) 0.33

1.00(l) 1.21(l)

0.64(l) 0.75(l)

1.00(l) 0.16 0.08 1.10(l) 1.27(l)

1.17(l) 0.52(2)1 0.83(l)

0.96(l) 0.80(l) 1.080) 2.38(2)

1.00(l) 0.10 0.80(l)

6 61

-5.0 20.3

Blue PC

0.62(l) 0.86(l)

15 11 0 3.0 Blue PC

0.79(l) 1.30(l) 5 7 30 7

-4.5 -1.5 24.5 24.5

Blue Blue PC PC

2 2 4 2 3 9 2 3 11 12 10 22 20 33.2 84 3.0

-9.5 -7.2 -5.5 -8.5 -10.5 -3.0 -11.5 -8.5 6.0 16.7 2.5 3.0 1.8 16.0 3.0 4.0

Yellow Blue Blue Blue Yellow Heme Blue Yellow PC PC PE PE PE S, PE S, PE S

T-VIII<

- - -

0.96(1

1.03(l)

0.90(l) 1.08(l)

-

1.05(l)

0.65(l)

(2) t

* Identified &s free lysine in Peptide T-V and &s Lys-Lys in Peptide T-XIIIb by paper chromatography. t The recovery of half-cystine in this analysis of the heme peptide is low. The assumed value of 2 is taken from the data of Tuppy and P&us (10).

decision as to which of the first 2 aspartyl residues is present as the free acid. Fragment P-l, obtained by papain digestion, is acidic, and since its composition corresponds to that of the 3 amino-terminal residues, the free aspartic acid must be the resi- due in position 50.

Residues 55 to 59: Lys-Gly-Ile-Thr-Trp (Table VI, Peptide C-Xllb)-Peptide C-XIIb is the only tryptophan-containing peptide recovered, and its mobilities correspond exactly to those of the single Ehrlich reagent-positive spot observed on the peptide map of the original chymotryptic digest of the protein. The protein thus contains only a single tryptophanyl residue.

On the assumption, from chymotryptic specificity, that trypto-

phan is carboxyl-terminal, three steps of Edman degradation were sufficient to established the sequence given.

Residues 60 to 65: Gly-Glu-Asp-Thr-Leu-Met (Table VII, Peptide C-I)-Since carboxypeptidase A digestion of Peptide C-I yielded equal amounts of methionine and leucine, while extensive chymotryptic digestion gave free methionine and a residual peptide, the carboxyl-terminal sequence of the parent peptide must be Leu-Met. Three steps of Edman degradation of Peptide C-I established the sequence listed above. Com- plete leucine aminopeptidase digestion of Peptide C-I released no trace of amidated residues as identified by paper electro- phoresis and chromatography.

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514 Chicken Heart Cytochrome c Vol. 241, h-o. 2

Residues 66 to 67: Glu-Tyr (Peptide C-VIb)-This dipeptide (Glu, 1.05(l); Tyr, 0.95(l); el, $14.6; ch, 23.0) was recovered in 30y0 yield after purification by paper electrophoresis followed by paper chromatography. Its structure is assumed from chy- motryptic specificity.

Residues 68 to 74: Leu-Glu-Asn-Pro-Lys-Lys-Tyr (Table VIII, Peptides C-X6 and C-XIa)-Both the peptides represent- ing this area are basic, requiring at least 1 of the acidic residues to be amidated. Leucine aminopeptidase digestion of Peptides C-XIa and C-Xb liberated only glutamic acid, and glutamic acid and leucine, respectively, indicating the amino-terminal sequence to be Leu-Glu. These results further indicated that proline is in position 71 (21) and that the aspartyl residue is amidated. The compositions of the two tryptic fragments from Peptide C-XIa, T-l and T-2, and two steps of Edman degradation on T-l established the sequence given, assuming from chymotryptic specificity that tyrosine is carboxyl-terminal in the parent peptide. It may be noted that since lysine is the carboxyl-terminal residue of T-l, the two steps of Edman degradation of this peptide also fix the position of the proline.

Residues 75 to 80: Ile-Pro-Gly-Thr-Lys-Met (Table IX, Peptide C-Vc)-Tryptic digestion of Peptide C-Vc yielded free methionine and a basic peptide (el, -4.0; ch, 9.5), indicating that the carboxyl-terminal sequence is Lys-Met. Three steps of Edman degradation were sufficient to establish the structure given above, the threonyl residue being placed before the lysyl residue by difference.

Residues 81 to 82: Ile-Phe (Peptide C-Vb)-Peptide C-Vb (Be, 086(l); Phe, 1.15(l); el, 0; ch, 36.5) was recovered in 22% yield. Chymotryptic specificity requires the sequence to be Ile-Phe.

Residues SS to 87: Ala-Gly-Ile-Lys-Lys (Peptide C-XIIa)- Peptide C-XIIa (Ala, 0.96(l); Gly, 0.99(l); Ile, 0.89(l); Lys, 2.16(2); el, -12.3; ch, 2.7) was recovered in 4y, yield. Three steps of Edman degradation established the sequence given, as follows.

Step I: Ala, 0.10; Gly, 1.00(l); Ile 1.20(l); Lys, 1.80(2). Step II: Ala, ~0.10; Gly, ~0.10; Ile, 0.80(l); Lys, 1.50(2). Step III: Only Lys remaining. Residues 88 to 94: Lys-Ser-Glu-Arg-Val-Asp-Leu (Table X,

Peptides C-VIIa and C-Xa)-Carboxypeptidase A digestion of Peptides C-VIIa and C-Xa released leucine and a small amount of aspartic acid in both cases, indicating a common Asp-Leu carboxyl-terminal sequence. Therefore, the extra lysyl residue in Peptide C-Xa must be amino-terminal.

The amino acid compositions, the electrophoretic mobilities, and one step of Edman degradation on each of the two tryptic fragments, T-l and T-2, recovered from Peptide C-VIIa de- termined the sequence of the fragments as well as the presence of glutamine in T-l and aspartic acid in T-2. The relative positions of the two tryptic fragments are obvious from the known carboxyl-terminal sequence of the parent peptide.

Residues 95 to 97: Ile-Ala-Tyr (Peptide C-IV)-Peptide C-IV (Ile, 1.12(l); Ala, 0.78(l); Tyr, 1.10(l); el, 0; ch, 27.5; yield, 41%) was subjected to two steps of Edman degradation, yielding the above sequence.

Step I: Ile, 0.10; Ala, 0.99(l); Tyr, 1.01(l). Step II: Ile, 0.10; Ala, 0.20; Tyr, 1.00(l). Residues 98 to 104: Leu-Lys-Asp-Ala-Thr-Xer-Lys (Table

XI, Peptides C-VIII and C-IXa)-The amino-terminal residue of Peptide C-VIII is leucine, as determined by Edman degrada-

tion. Following extensive tryptic digestion, only two fragments were obtained. It is important to note that no free lysine could be detected, indicating that the peptide did not contain a Lys- Lys sequence. T-l must be Leu-Lys from tryptic specificity and because t,he single leucine in the parent peptide is amino- terminal. Three steps of Edman degradation gave the sequence of the carboxyl-terminal fragment T-2, the carboxyl-terminal segment of which must be Ser-Lys, since if the position of these residues were inverted the seryl residue would not have been recovered in T-2 and the tryptic digest would have con- tained a third fragment, free serine. The carboxyl-terminal position of lysine was confirmed by hydrazinolysis.

Amino Acid Compositions of Peptides from Tryptic Digest

The amino acid compositions of the peptides isolated from the tryptic digest of chicken heart cytochrome c are listed in Table XII. Since it is difficult to obtain complete digestion of the protein with trypsin (see “Discussion”), a relatively high con- centration of the enzyme was used and the hydrolysis was con- tinued for as long as 9 hours, as compared to 2% enzyme con- centration and a 3-hour digestion period, which were found to be satisfactory for the cytochrome c from the moth, S. Cynthia (9). It is therefore not surprising that some chymot.ryptic-like cleavages were observed, notwithstanding the prior treatment of the enzyme preparation according to Redfield and Anfinsen (12) to destroy contaminating chymotrypsin.

DISCUSSION

Enzymic Digestion of Cytochrome c-Complete proteolytic digestion is much more difficult to achieve with chicken cyto- chrome c than with either the moth (9) or the yeast (8) proteins. Thus, for example, conditions of proteolysis with trypsin (2% enzyme by weight, 3 hours at 38”) that yielded complete diges- tion of moth cytochrome c resulted in only a limited digestion of the chicken protein, as indicated by peptide maps. The more drastic conditions which had to be employed with the chicken protein yielded a number of probable chymotryptic cleavages, as can be seen in Table XII. However, only two peptides in the chymotryptic digest were in all probability the result of the tryptic activity, Peptides C-XIII and C-XIIa. It is interesting to note that the ready proteolytic digestibility of the yeast pro- tein is only one of the many lines of evidence indicating a looser structure, as compared to cytochrome c of vertebrate origins (1).

Even though the yields of the tryptic peptides were generally satisfactory, the yields of the chymotryptic peptides reported in the present paper are low. This was probably due t.o an error in the preparation of the resin used in the column chromatog- raphy, which resulted in the presence of sodium acetate in the peptide fractions and caused major losses in all paper purifica- tion procedures. Two areas which yielded particularly small amounts of peptides are the one immediately following the heme (Peptide C-XIII) and the one in the region (Peptide C-XIIa) preceding the Lys-Lys-Lys sequence (residues 86 to 88). In the former case, the major amount must have remained attached to the heme peptide which was not recovered, since the cleavage liberating Peptide C-XIII resulted from tryptic hydrolysis at lysyl residue 22. In the latter case, it is probable that the major peptide covering residues 83 to 94, because of its excessive positive charges, was not eluted from the Dowex 50-X8 column. In this connection it may be noted that the similar peptide

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Issue of January 25, 1966 X. K. Chan and E. Margoliash 515

from the horse protein came off Dowex X2 columns (20) only at the very end of the chromatographic runs.

Amino Acid Sequence of Chicken Heart Cytochrome c-The amino acid sequences of the chymotryptic peptides and the amino acid compositions of the tryptic peptides isolated from the corresponding digests of chicken heart cytochrome c are re- ported in Tables II to XII. The only segment of the protein for which no structural evidence is given in the present paper is the area covered by the heme peptide, the amino acid sequence of which is taken from the work of Tuppy and Paleus (10). The data taken as a whole are sufficient to establish the unique structure of the polypeptide chain given in Fig. 3. There is no necessity to discuss in detail the overlapping arrangements of chymotryptic and tryptic peptides which lead to the assign- ments of the relative positions of these fragments of the protein, since the arguments have been fully reported for several other cytochromes c, from essentially similar or identical peptides

(2, 3, 9). The amino acid sequence of chicken heart cytochrome c given

in Fig. 3 accounts for the total composition of the protein, de- termined directly on acid hydrolysates. However, a recently reported analysis of chicken heart cytochrome c (22) differs from that listed in Table I in that it shows 2 fewer residues of threonine and 1 fewer residue each of serine, methionine, and tyrosine, and adds up to a total of only 99 amino acids. It does not seem possible to account for the discrepancy except to sug- gest that the missing residues are exactly those which are ex- pected to undergo a relatively large extent of destruction during acid hydrolysis. It is unlikely, moreover, that a cytochrome c would have only 99 residues, since the shortest chain length so far reported, among more than 20 such proteins (l), is 103 amino acids (6, 7).

Comparative Structures of Cytochrome c-Chicken heart cyto- chrome c bears all the characteristic primary structure features of other “mammalian-type” cytochromes c. It consists of a single polypeptide chain, with a thioet.her-bonded heme prosthetic group located near the amino-terminal end of the chain. It has the typical cluster of hydrophobic and basic residues, and in general shows an amino acid sequence which is unequivocally recognizable as that of a cytochrome c (1). In fact, it differs by no more than 11, 12, 9, 9, 19, 28, and 45 residues from the horse (2), human (3), beef (4), pig (5), tuna (6, 7), moth (9), and yeast (8) proteins, respectively. Like other vertebrate cytochromes c, the chicken protein carries an acetylated amino- terminal residue. The evolutionary and functional implica- tions of variable and constant aspects of the primary structure of cytochrome c have been discussed previously (1) and will not be considered here.

Among the unusual residue substitutions observed in the chicken protein are the isoleucine, the glutamic acid, and the lysine in positions 3, 44, and 104, respectively. The isoleucine replaces a valine common to all other cytochromes c so far de- scribed. The glutamic acid replaces a proline common to most proteins of the group, except for tuna (6, 7) and yeast (8) cyto- chromes c, which also carry a glutamyl residue in this position.

The possible structural significance of the dispensability of the proline in position 44, as compared to the strict conservation of the other 3 common prolines in cytochrome c sequences (positions 30, 71, and 76), has been considered elsewhere (1). The 4 carboxy-terminal residues of cytochrome c have shown an ex- treme degree of variability in proteins from different species (1). The three cases so far reported in which a lysyl residue is the carboxyl terminus of the polypeptide chain are the cytochromes c from the chicken, the moth S. Cynthia (9), and iso-2-cytochrome c of bakers’ yeast (23). Whet.her this is a random phenomenon or represents distinct evolutionary lines cannot at present be assessed.

Acknowledgments-The authors are grateful to Robert Gar- disky for the amino acid analyses, and to Otto Walasek for the preparation of cytochrome c.

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S. K. Chan and E. MargoliashcAmino Acid Sequence of Chicken Heart Cytochrome

1966, 241:507-515.J. Biol. Chem. 

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