proteins of wheat bran. wheat has been from time immemorial and continues to be

PROTEINS OF WHEAT BRAN.

I. ISOLATION AND ELEMENTARY ANALYSES OF A GLOBULIN, ALBUMIN; AND PROLAMINB.*

BY D. BREESE JONES AND C. E. F. GERSDORFF.

(From the Protein Znvesligalion Laboratory, Bureau oj Chemistry, United States Department of Agriculture, Washington.)

(Received for publication, August 30, 1923.)

Wheat has been from time immemorial and continues to be probably the most important item in the food of civilized man. The proteins of the wheat germ, and those proteins of t,he endosperm which collectively are commonly known as gluten, have been extensively investigated by Osborne and coworkers and others. We have but little knowledge, however, of the proteins contained in the milling by-product of wheat commonly called bran.l

On account of the thick walls of the cells in the layer containing the proteins, the extraction of the proteins from the bran has been considered so difficult that they have never been isolated in sufficient quantity or suitable condition for a study of their chemical or phvsical character. Furthermore, even the cleanest grades of commercial wheat bran always contain nn admixture of an appreciable quantity of starch and gluten, which are removed with difficulty.

Bran is extensively used in the feeding of farm animals and has been long regarded as having a high nutritive value. During recent years there has been an increasing recognition of the

* A preliminary report of this paper was presented at the meeting of the American Chemical Society held in New Haven, Conn., April 2 to 9, 1923.

1 The term “bran” is used in this paper to include the outer seed coats, together with the underlying layer of cells which contains the protein, and which in the milling process is removed with, and is firmly attached to, the outer seed ooats.

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118 Proteins of Wheat Bran

value of bran as an element in human food. Commercial methods are now perfected for the prepara.tion of bran as a palatable cereal breakfast food.

It is generally known that the nutritive value of so called “patent” wheat flour is inferior to that of whole wheat, being deficient in vitamins, inorganic constituents, and defective in the quality of its proteins. It therefore appears very probable that the proteins of the bran are of a different chemical nature, and contain satisfactory quantities of those amino-acids which are deficient in the gluten proteins. Although the results of elementary analyses of the proteins reported in this paper support the above supposition, we do not wish to draw any final conclusions as to the identity or non-identity of the bran proteins with the corresponding proteins of the other parts of the wheat kernel, until we have obtained further knowledge of the amino-acid composition of the bran proteins. Experiments to this end are now in progress.

In their classic work on “The prot,eins of the wheat kernel.” Osborne and Voorhees2 have shown that the proteins of wheat gluten, which comprises the proteins of white, wheat flour, consist chiefly and in about equal quantities of gliadin, t,he alcohol- soluble protein, and glutenin, a protein which is insoluble in water, neutral salt solutions, or alcohol, but is soluble in dilute acids or alkalies. The proteins of the wheat embryo as shown by Osborne and Campbell3 differ dist,inctly in character from those of the gluten, and consist of globulin, albumin, and prot,eose. These proteins, however, constitute but a relatively small proportion of the total protein of the wheat kernel.

The composition of bran varies with the kind of wheat and the locality in which it is grown. The following composition of a sample of commercial bran is given by Osborne and Mendel.4

Composition of Commercial Bran, Moisture-Free. per cent,

Sucrose................................................... 1.64 Dextrin.................................................. 4.19 Starch.................................................... 13.39

* Osborne, T. B., and Voorhees, C. G., Am. Chem. J., 1893, xv, 392. 8 Osborne, T. B., and Campbell, G. F., J. Am. Chem. Sot., 1900, xxii, 379. 4 Osborne, T. B., and Mendel, L. B., J. Bid. Chem., 1919, xxxvii, 657.


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13. 13. Jones and C. E. F. Gcrsdorff

Pentosnns, etc.. . . . . . . . ...... 27 SY Protein (N X 6.25) _. . . . . . ...... ...... 17 .OO Fiber . . . . . . . . . . . . . . . . . . . . . . . . ...... . . ...... 9.37 Fat. . . . . . . . . . . . . . . . . . . . . . . . . . ...... ...... 7.07 Ash.......................... . ....... 7.24 I’ntlctcrmined.. . . . . . ...... 12.52

lnn.nn

Rrnn also contains rchttivtlg large qwntitics of inorg:wic salts. Jortlan, Hart, ant1 Etttcn” give thr: following :mnlyscs.

P = 1.42, (::I ns CnO = O.lS, Rig RS AI& = O.r;9, l\ xi l<& = 1.5s

The npprosim:lte proportion of cndospcrm, bran, and cm- lqo in the mhwt kernel, the proportion of the total protoiri of the sect1 in thcsc parts xntl ;dso tho pcrwntngct of the total protein in the part, itself, is givctn in 0x2 nccomp:inying t.ubulation by Osborne and Mcndcl:~

Endosperm. . .

1. -..I-.

s3.5 1 .Gl l3rnn.. . . . . . . . 15 .o 0.40 FImbrgo.. . . . . . . . . . .; 1.5 1 0.10

I

100.0 : 2.20

‘J.li 11.1 X3.3

2.so 18.7 23.3 0.57 3G.i 4.4

12.5-L 100.0

In view of the little that is known rcgnrding the proteins of wliwt brim, its rchltivcly high content of protein, :1ritl of its nutritional and economic importnnw, WC have ~~ntlcrt:~l;cn an invcstig:ltion of the proteins of wheat, br:1n with the view of their isolntion and the dctc~rminnt ion of t.hcir ph~~r;ic:ll :mtl chcmi- cd characters, :wl of obtaining such informxt ion conwrriing the inclivid~~d proteins of bran as WC :~lrcnd~ have rc~g:lrtling the proteins of the othw parts of t.1~ wheat ktwcl.

In t.his pnpcr :1rc (Icscrihctl tllc prcp:ir:il ion an[l propcrtics of three protc+is which h:lvc bwn isol:~tcd from who:1t bran so trcntcd as to rcmo\‘c most of the otlwr parts of the sectl which


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are usually found associated with commercial bran. The proteins isolated consisted of an albumin, a globulin, and a prolamine.

The isolation and preparation of proteins to be used for a study of their properties and composition require that they be obtained in as pure a state as possible. This calls for methods of purificat.ion, such as reprecipitations, repeated washings, and filtration-processes which frequently involve large losses of material. Furthermore, in many cases the extractions of the

TABLE I.

Proteins Extracted and Isolated from Wheut Bran.

Total protein in the bran (N X 6.25 = 17.25 per cent).

Nitrogen extrmted (per cent of bran).

--

Extracted by distilled water.. . . . . . . 0.63 1. Albumin....................... 2. Globulin.. . . . . . . . . . . . . . . . . . .

Extracted by 4 per cent sodium chloride......................... 0.35

Globulin. . . . , . . . . . . . . . . . . . . . . . . . . . Extracted by 70 per cent alcohol. . . 1 .Ol

Prolamine......................... Extracted by 0.5 per cent sodium

hydroxide. . . . . . . . . . . . . . . . . . . . . . . 0.40

Total.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Protein extraated NX0.20 (per cant of bran).

3.94

2.19

6.31

2.50

T

Protein isolated.

2.87 0.69

1.66

5.35

16.64 4.00

9.62

31 .Ol

61.27 -

l Equivalent to 86.61 per cent of the calculated total protein in the bran.

proteins from their parent substances have been incomplete, the object having been merely to secure sufficient quantities of the purified proteins for their chemical study. Consequently, the yields of the proteins aa finally obtained oftentimes do not nearly represent the quantities in which they are present in substances from which they were obtained. To know as nearly m possible the percentages of the different proteins present in a given seed or foodstuff is frequently of great importance. In order to ob- tain as accurately aa possible the percentages of the different proteins present in wheat bran, successive exhaustive extractions of


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D. B. Jones and C. E. F. Gersdorff 121

the washed bran meal were made with distilled water, 4 per cent aqueous sodium chloride solution, boiling 70 per cent alcohol, and with 0.5 per cent sodium hydroxide. The proteins removed by these various solvents were separated and isolated by methods involving minimum losses. The yields found are con-

TABLE II.

Average Results of Duplicate Analyses of Wheat Bran Albumin.+

Preparatmn.

I II III IV --~-

per cent per cent per cent per cent

C . . . . . . . . . . . . . . . . . . . . 53.20 53.25 53.27 53.29 H . . . . . . . . . . . . . . . . . . . . 6.79 6.77 6.77 6.47 N . . . . . . . . . . . . . . . . . . . . 15.09 15.51 15.28 15.61 S . . . . . . . . . . . . . . . . . . . . 1.24 1.39 1.38 1.43 0 . . . . . . . . . . . . . .._... . 23.68 23.08 23.30 23.20 Moisture. . . . . . . . . . . . . 10.24 5.08 3.95 4.40 Ash.. . . . . . . . . . . . . . . . . 1.49 0.65 0.29 0.41

* Calculated on an ash- and moisture-free basis.

TABLE III.

V Average.

pm cent 53.05 6.75

15.61 1.33

23.26 4.66 4.59

per cent

53.21 6.71

15.42 1.35

23.30

Average Results of Duplicate Analyses of Wheat Bran Globulin.*

Preparation.

I II III IV v VI VII AWP age.

-- ------ percent percent percent pet cent percent percent percent percent

C . . . . . . . . . . . . . . . . . . . 53.41 53.49 53.58 53.37 53.59 53.41 53.19 53.43 H . . . . . . . . . . . . . . . . . . 7.68 7.69 7.37 7.35 7.30 734 7.10 7.40 N . . . . . . . . . . . . . . . . . . . 17.94 17.89 17.81 18.01 17.67 17.39 L7.59 17.76 S . . . . . . . . . . . . . . . . . . . 0.91 0.92 0.90 0.92 0.89 0.90 0.90 0.91 0 . . . . . . . . . . . . . . . . . . . 20.06 26.01 20.34 20.35 20.55 20.96 21.22 20.50 Moisture.. . . . . . . . . . . 10.11 9.20 4.70 5.16 10.33 5.01 4.16 Ash. . . . . . . . . . . . . . . . . 0.60 1.25 0.24 0.36 1.49 1.81 0.17


siderably higher than those obtained in the preparation of sam- ples for the analytical study of the proteins, and, we believe, quite closely represent the quantities in which these proteins were present in the bran.


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122 Proteins of brhest Bran

Ry cxhaustivcly cxt ratting the bran with the solvents used, there was rcmovcttl an amount of nitrogen cquivalcnt to Ni.Gl

per cent of tlic: c:A~ulnt.ctd (PI; X G.25) total protein in the bran. E’rom t.hcsc extracts, there were isolated the following pcrccn- tngcs of proteins, cxprcsxcd in terms of t.lic bran used: albumin, 2.8i per writ; globlilirt, 2.35 per cent; and prolamine, 5.35 per cent. The results of thcsc extraction experiments arc given in Table I.

Scvcr:d prcparntions of cacti of the three proteins isolated from bran wrc matlc by different methods. It is of intcrcst. to note the close agrcctmcnt of the results obtained on analyses

TABLE IV.

Average Results of Dup1ica.k Annlyscs OJ’ the Alcohol-Soluble Protein Jrom Wheat Bran.*

- I’rcpuration.

II III IV - I:.

) cr cent I’W rent per <C,ll / prr crnl

54.05 54.20 54.38 / 54.30 6.i4 ! G.iS 6.64 G.87

15.56 15.34 : 15.42 15.21 1.26 1.37 1 1.37 , 1.35

-- 1.

22.39 ( 22.31 ! 22.19 i 22.2.1 j 22.30 hloisture . . . . . . . . . .._ ,’ Y.00 ; 8.24 Ash . . . . . . . . . . . . . . . . . . 0.M 1 0.46


of the same protein obtained by different mct.hods. (Tables II, III, and Jv.) The composition of thcsc proteins differs from that of similar proteins obtained by Osborne and Yoorhccs’ from “shorts,” a commercial product, consistirl,q largely of bran but also containing cot~sidcrnblc portions of the starchy cndo- sperm of the seed.

Frqmrahu oj illa~erial.--Thc bran used for t,hc csperimcnts described in this paper was furnished by the TIcckcr-Jones- Jcwcll Milling Company, and was obtained most.ly from 7.5 per cent I)ark 1 Northern Spring Wheat and 23 per cent of 2 IIard Kansas Wheat. Previous to grinding, the wheat had hvn tcm-


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D. B. Jones and C. E. F. Gersdorff

pered by the addition of water and passed to bins at room temperature, where it remained for 8 hours before grinding. Addi- tion of moisture before milling is made to toughen the bran so as to facilitate its removal from the other parts of the kernel. Com- mercial bran always con.tains appreciable quantities of adhering parts of the rest of the kernel. In order to remove as much as possible of this adhering material, the bran was placed in cheese-cloth and rapidly washed by vigorous plunging in cold t.ap water. By this method most of the adhering starchy endosperm was removed. The turbid washings were reserved for later examination. Immediately after washing, the bran was dried at 30°C. in a motor driven fan drier, and ground so as to pass through a 100 mesh sieve. The washing and drying caused the bran to lose much of its t,ough character, making it possible to grind it more easily. Preliminary extract*ions of t,hc bran with protein solvents showed, however, that equally efficient extractions of the protein could be obtained from bran which had been ground to pass through a 40 mesh sieve.

Microscopical examinations of both the unwashed and washed bran were made by J. F. Clevenger, of the Pharmacognosy Laboratory of this Bureau. It was estimated that at least 75 per cent of the starchy endosperm in the original bran had been removed by the washing, and that the resulting washed and dried bran contained less than 5 per cent of material other than bran. The presence of embryonic tissue in the original bran could not be detected. Analysis of the washed bran showed a content of 3.6 per cent of starch. This percentage is in fair agreement with the estimate based on the microscopical examination. Since the ratio of protein to starch in wheat endosperm is approximately l:f, as pointed out by Osborne and Mendel,* the amount of endosperm protein in our washed bran can be estimated from its starch content at approximately 0.5 per cent. It is not believed, however, that this small quantitv of protein derived from the starchy endosperm is sufficient to cause any apprecia,ble error in the data presented in this paper on the proteins of the bran.

Total nitrogen determinations, calculated on an ash- and moisture-free basis, showed that the unwashed bran contained 2.84 per cent of nitrogen, or 17.75 per cent of protein (N x 6.25),


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and that the washed bran contained 2.76 per cent of nitrogen, equivalent to 17.25 per cent of protein.

Exantination of the Water Washings from the Bran.-The turbid washings of the commercial bran, obtained as previously described, were filtered clear and examined for the purpose of ascertaining the quantity and the nature of the protein removed by the washing. Gradual heating of the filtered washings after slightly acidifying with 1 per cent acetic acid, caused the separation of a flocculent coagulum at 55°C. From the entire washings, there was isolated of this albumin 0.3 per cent of the bran used. It had the following percentage composition: Carbon, 53.05; hydrogen, 6.90; nitrogen, 16.64; sulfur, 1.33 per cent.

The composition of this protein agrees more closely with that given by Osborne and Voorhees2 for the albumin isolated by them from “straight wheat flour” than it does with that of the bran albumin described in this paper (Table II). Moreover, the coagulation point (55°C.) of the albumin obtained from the washings agrees with that given by Osborne and Voorhees2 for the albumin they isolated, while the albumin obtained from the washed bran coagulated at 65°C. These considerations indi- cate that the protein in the water washings came chiefly from adhering portions of other parts of the seed, rather than from the protein cells of the bran itself.

Exhaustive Extraction of the Bran Meal with Various Solvenis.

Preliminary extra&ions of small quantities of bran, ground to pass through a 40 mesh sieve, were made to determine the most suitable conditions, such as concentration of solvents, time of extraction, and temperature, for the most efficient extraction of the protein from the bran. 4 per cent aqueous sodium chloride solution was found to be the most eilkient concentration of this salt. The results of these experiments are given in Table V.

Extraction with Distilled Water.-The bran meal was exhaustively extracted by distilled water at l-2°C. The extractions were made at low temperature so as to avoid danger of fermenta- tion, to which wheat bran is quite susceptible. The extractions were continued until practically nothing was being removed by the distilled water. The clear, filtered extracts were then dia-


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D. B. Jones and C. E. F. Gersdoti 125

lyzed in running chilled water for 12 to 14 days. This caused the separation of a small quantity of globulin, which amounted to 0.69 per cent of the meal extracted. The filtrate from this globulin was saturated with carbon dioxide gas, which caused the further separation of a trace of globulin which was removed by filtration. The filtrate was then boiled until coagulation of

TABLE V.

Extraction Experiments.*

Preliminary extrscti0ns.t

1.0 per cent NaCl.. .... 2.0 “ “ “ ...... 3.0 “ “ “ ...... 4.0 (( (‘ Cc ...... 7.0 ‘( (( . “ ......

10.0 “ “ ‘( ...... Distilled Hz0 ........... 70 per cent alcohol ......

per cent

. 3.25 1

. 4.81 2

. 4.94 3

.5.00 4

.3.94 5 3.75 6

. 1.13 7

. 3.69 8

I Time extraations.~

so1vent.t

4 per cent N&l, 1 hr. 4 “ ‘( “ t “ 4 ‘L “ “ 1 “ 4 ‘< ‘< “ 4 hrs. 4 “ “ “ 5 “ 4 “ “ “ 6 “ Distilled &O, 20 “

“ “ 66 (‘

A per cent

3.50 3.50 5.00 5.66 5.25 5.94 2.13 3.94

* All extractions were made on fresh material not previously extracted with any other solvent.

t These extractions were carried out at room temperature for a period of 1 hour each with frequent stirring.

1 Solvents were used in the proportion of 20 cc. per gm. of meal. 0 These extractions were carried out at room temperature with frequent

stirring except the last two which were held in cold storage at l-2°C

the albumin was complete. The albumin thus obtained amounted to 2.87 per cent of themeal, or 16.64 per cent of the total protein of the meal.

The filtrate from the coagulated albumin was concentrated, and 2 volumes of 95 per cent alcohol were added, where upon a precipitate amounting to 1.49 per cent of the meal separated. This precipitate contained but a small quantity of nitrogen and may have consisted of a small amount of proteose mixed with other impurities.


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Proteins of Wheat Bran

Extra&ion with 4 Pq Cent Sodium Chloride Solution.--The residue remaining after the extractions with distilled water was next extracted with 4 per cent sodium chloride solution at room temperature. Nitrogen determinations made on aliquot portions of the extracts showed that exhaustive extraction with the salt solution removed 0.35 per cent of nitrogen, equivalent to 2.19 per cent of protein. The joint saline extracts were acidified with acetic acid, and boiled until there was no further separation of coagulum. The precipitate was collected on a filter, washed with distilled water, and dried at 110”. The yield of &dJdin thus obtained was 1.66 per cent of the meal, or 9.62 per cent of the total protein in the bran.

Extraction with 70 Per Cent Alcohol.-The residue remaining after the extraction with sodium chloride solution was cxhaus- tively extracted with boiling 70 per cent alcohol. The alcohol removed 1.01 per cent of nitrogen, equivalent to 6.31 per cent of protein. The joint extract,s were filtered and concentrated under reduced pressure until most of the alcohol was removed. The flocculent precipitate, which separated as the alcohol was being removed, was allowed to settle and was washed by dccan- tation with water and dried at 110°C. It amounted to 5.35 per cent of the meal, or 31.01 per cent of the total protein in the meal based on N x 6.25.

Extraction. with 0.5 Per Cent Sodium Hydroxide Solution.- The residue from the alcohoi extractions was then extracted with 0.5 per cent sodium hydroxide at room temperature. The alkaline extracts contained 0.40 per cent of nitrogen, equivalent to 2.50 per cent of protein. The joint extracts were acidified with acetic acid, and several volumes of alcohol were added, which cauied the precipitation of a substance containing but a small amount of nitrogen.

Qualitative tests made on this substance for phytin and purines gave negative results, neither did it respond to the Riuret, gly- oxylic, or Millon tests for protein. The material ignited readily, leaving a large ash residue.

Exhaustive extractions with these four solvents therefore removed nitrogen equivalent to 14.94 per cent of protein, calculated on the basis of the bran used. This is equivalent to 86.61 per cent of the calculated total protein in the bran. There


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were actually isolated as identified proteins, 2.35 per cent of globulin, 2.87 per cent of albumin, and 5.35 per cent of prolamine-a total of 10.57 per cent of the bran or 61.27 per cent of the calculated total protein in the bran. The results of these exhaustive extractions are summarized in Table I.

Since probably not all the nitrogen in bran is protein nitrogen, the actual quantit,y of protein present would therefore be less than the 17.25 per cent obtained by multiplying the nitrogen by 6.25. This would give a correspondingly higher value for the percentage of protein actually isolated than the 61.27 per cent here reported.

Preparation of the Proteins.

All the preparations described and analyzed, excepting one, were obtained by successive extractions of each sample of the washed bran meal by the various solvents.

The Albumin.-The albumin preparations were obtained by the following methods.

1. A distilled water extract of the bran was dialyzed for 10 days against chilled rubning water a.nd the dialysate saturated with washed carbon dioxide. The small quantity of precipitated globulin was removed by filtration, and the filtrate wa.s heated for 30 minutes at 65°C. The coagulated protein was thoroughly washed with distilled water and dried in the usual way with alcohol and ether. The yield was 0.1 per cent (Preparation I).

2. Preparation II was obtained in a 0.26 per cent yield by dialysis of a 4 per cent sodium chloride extract against chilled running water for 12 days, and subsequent treatment of the dialysate as given for Preparation I.

3. A 4 per cent sodium chloride extract was saturated with ammonium sulfate and the precipitated protein redissolved by addition of distilled water. The solution was filtered and dialyzed for 16 days. After removal of the globulin, the dialysate was then treated as in the two previous preparations (Prepara- tion III). A yield of 0.18 per cent was obtained.

4. A distilled water extract without previous dialysis was saturated with washed carbon dioxide, and then slightly acidified with 1 per cent acetic acid. The precipitated globulin was re-


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moved by filtration, and the albumin coagulated by heating the filtrate for 30 minutes at 65°C. The albumin, after washing and drying in the usual way, was obtained in 0.99 per cent yield (Preparation IV).

5. A 4 per cent sodium chloride extract of the bran was saturated with washed carbon dioxide gas. This caused but an incomplete precipitation of the globulin. The remainder was separated by slowly adding, drop by drop, dilute acetic acid, the end-point being indicated by the flocculent character of the precipitated protein. After removd of the globulin by filtration, the albumin (Preparation V) was coagulated, washed, and dried, as in the case of the previously described preparations (yield, 2 per cent).

The average results of duplicate analyses of these five albumin preparations, calculated on an ash- and moisture-free basis, are given in Table II.

The Globulin.-The globulin preparations were obtained from a 4 per cent aqueous sodium chloride extract of the bran meal. Seven preparations were made by various methods. In all cases the precipitated proteins were finally washed and dried in the usual way.

1. Preparation I was obtained in 0.63 per cent yield by making the extract 0.55 saturated with ammonium sulfate. The precipitate was removed by filtration, redissolved by addition of water, and the solution was filtered. The globulin was then precipitated from the clear filtrate by dialysis.

2. Preparation II was made in the same way as Preparation I, with the exception that the extract was made 0.65 saturated with ammonium sulfate. Yield, 1.0 per cent.

3. By complete saturation of the extract with ammonium sulfate, and subsequent removal of the globulin by dialysis, Preparations III and IV were obtained in 0.97 and 0.53 per cent yields.

4. Preparations V and VI were obtained by directly dialyzing the salt extract against chilled running water for 12 days.

5. Acidification of the extract with carbon dioxide, followed by the addition of dilute acetic acid, as described under the preparations of the albumin (Method 5), yielded Preparation VII in 2 per cent yield. Average results of the analyses of t,hese preparations are given in Table III.


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D. B. Jones and C. E. F. Gersdorf? 129

The AlcohoLYoluhle Protein.-Five preparations of the alcohol- soluble protein, made in several ways, were obtained as follows: Preparations I to IV, inclusive, were isolated from bran meal which had been previously exhaustively extracted with distilled water and 4 per cent sodium chloride solution. This residue was extracted with boiling 70 per cent alcohol for 15 minutes, and the hot mixture was immediately filtered. Preparation I was obtained by pouring the extract into a 4 per cent sodium chloride sol&ion. The precipitate was thoroughly washed with water and dried in the usual way. The yield was 1.0 per cent of the original bran used before the extraction with water and salt.

Concentration of the alcoholic extract under diminished pressure until most of the alcohol had been removed caused the separation of the prolamine in a form which was no longer soluble in alcohol. This precipitate constituted Preparation II. The yield was 1.7 per cent.

Preparation III was isolated from a highly concentrated alcoholic solution of the protein. This solution was obtained by occasional addition during the distillation under diminished pressure of small quantities of 95 per cent alcohol, so as to pre- vent precipitation of the protein due to the diminishing content of alcohol in the distillation reservoir. The protein was precipitated from the concentrated solution by addition of several volumes of absolute alcohol. After washing and drying the precipitated protein in the usual way, a yield of 4.29 per cent was obtained.

Preparation IV, obtained in 1 per cent yield, was isolated as in the case of Preparation III, with the exception that the product obtained by precipitation with absolute alcohol was redissolved in boiling 80 per cent alcohol and then reprecipitated by absolute alcohol, after having first Ntered off some undis- solved protein which had become denatured.

Preparation V was isolated from bran which had not been previously extracted with other solvents. The method of preparation otherwise was the same as that used for Preparation III. Because of an accidental loss of a portion of the extract used in this preparation only a small yield was obtained.

The elementary composition of the prolamine preparations is given in Table IV.


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Properties of the Proteins.

The Albumin.--The albumin preparations obtained as already described ranged in color from pale cream to gray.

In aqueous solution, slightly acidified with acetic acid, the albumin coagulated at 60°C. When heated in 4 per cent aqueous sodium chloride solution, it coagulated at 65”C., and at 62°C. in a 4 per cent sodium sulfite solution. Its coagulation point in water is somewhat raised by the presence of a small quantity of the globulin.

The Globulin.--Dilution of saline extracts of the bran with large volumes of water showed that the globulin was readily soluble in dilute aqueous sodium chloride solutions. Conse- quently, its preparation by the customary method of diluting the saline extract is impracticable. It can be precipitated in small quantities from a sodium chloride solution by saturation with carbon dioxide, and practically completely by slight acidification with acetic acid. The precipitate thus formed is readily soluble in 4 per cent sodium chloride solution.

The precipitation limits with ammonium sulfate ranged from 0.4 to 0.65 of saturation.

The globulin coagulates in a slightly acidified 4 per cent sodium chloride solution at 95°C.

The preparations were obtained in the form of dusty powders, ranging in color from white to cream.

The Alcohol-Soluble Protein.--This protein precipitated by absolute alcohol from a concentrated alcoholic solution is readily soluble in boiling 80 per cent alcohol. It differs from gliadin in its behavior when separating on cooling from warm 70 per cent alcohol. Much of t,his protein rapidly precipitates in a finely divided form, which redissolves on warming. Addition of aqueous sodium chloride to its alcoholic solution causes the prolamine to separate in flocculent particles which soon adhere to form a sticky precipitate. Addition of 2 volumes of 2 per cent hydro- chloric acid to the prolamine in 70 per cent alcohol produces a flocculent precipitate which redissolves on boiling and again separates on cooling.


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SUMMARY.

Three proteins, an albumin, a globulin, and an alcohol-soluble protein, have been isolated from wheat bran. The bran, which originally was exceptionally clean, was rapidly washed in cold water and immediately dried at a low temperature. In this way the greater part of the adhering particles of other portions of the wheat kernel was removed. The bran was then ground to a fine powder. This contained 17.25 per cent of protein (N x 6.25). By successive exhaustive extractions with distilled water, 4 per cent sodium chloride solution, 70 per cent alcohol, and 0.5 per cent sodium hydroxide solution, 86.61 per cent of the total protein in the bran was extracted (N x 6.25). The percentages of proteins actually isolated, expressed in terms of the total protein in the bran, were as follows: Albumin, 16.64; globulin, 13.62; alcohol-soluble protein, 31.01 per cent. Analyses showed these proteins to have the following average elementary percentage composition: Albumin, N 15.42, C 53.21, H 6.71, S 1.35; globulin, N 17.76, C 53.43, H 7.40, S 0.91; alcohol-soluble protein, N 15.35, C 54.25, H 6.75, S 1.35.


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D. Breese Jones and C. E. F. GersdorffALBUMIN; AND PROLAMINEANALYSES OF A GLOBULIN,

ISOLATION AND ELEMENTARY PROTEINS OF WHEAT BRAN: I.

1923, 58:117-131.J. Biol. Chem.

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proteins of wheat bran. wheat has been from time immemorial and continues to be

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