PROTEINS OF WHEAT BRAN.

II. DISTRIBUTION OF NITROGEN, PERCENTAGES OF AMINO ACIDS AND OF FREE AMINO NITROGEN: A COMPARI-

SON OF THE BRAN PROTEINS WITH THE CORRE- SPONDING PROTEINS OF WHEAT ENDOSPERM

AND EMBRYO.

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

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

(Received for publication, April 6, 1925.)

Approximately 22 per cent of the nitrogen of the wheat kernel resides in the seed coat,s or bran. This represents a vast amount of protein in the t.otal wheat, crop which is produced annually. Inasmuch as wheat is probably the most extensively used item in the food of civilized man, it is rather surprising that heretofore practically nothing has been known regarding the character and properties of the proteins of wheat bran. Results obtained by the use of bran in the practical feeding of animals have led to the general view that the bran proteins have a high nutritive value. It has been also amply demonstrated by feeding experiments with small animals that the proteins of the whole wheat kernel ade- quately supply Dhe animal’s dietary protein requirements, and that they are superior in this respect to the proteins of the endo- sperm, which constitute almost entirely the proteins of ordinary white wheat flour. From these considerations it appeared prob- able that the bran proteins are of such a character as to supple- ment nutritively those of the endosperm; that, they are rich in those nutritionally essential amino acids in whic’h the endosperm proteins are deficient. The results of our studies on the bran proteins have substantiated the above supposition.

The writers have previously shown (1) t,hat the proteins of wheat bran consist essentially of a prolamin, a globulin, and an albumin. These proteins were obtained in yields of 5.35, 2.35, and 2.87 per

241

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242 Proteins of Wheat Bran. II

cent, respectively, and were isolated from a hard wheat bran, from which nearly all of the other parts of the seed usually asso- ciated with commercial bran had been removed by a special treat- ment (1). The above percentages of protein which were isolated represent *collectively 61.27 per cent of the total protein in the bran as calculated from its nit.rogon content. Since not all of the nitro- gen of the bran is protein nitrogen, the percentage of the total true protein isolated must be somewhat higher than t.he above figure cited.

Although the elementary composition of the bran proteins and some of their properties strongly indicated that these proteins differed from the corresponding proteins of the wheat endosperm and embryo, final conclusions on this point were deferred until further knowledge regarding them should be available.

A striking difference between the general character of the pro- tein content of the wheat bran and that of the endosperm lies in the fact that approximately half of the protein of the endosperm consists of a glutelin (glutcnin), while,, on the other hand, no protein of the glutelin type was found in the bran.

In this paper we are now presenting data showing the dis- tribution of nitrogen and the percentages of the dibasic amino acids in the bran proteins as determined by the Van Slyke method. The tryptophane, cystine, and tyrosine contents of these proteins have also been estimated by means of calorimetric methods.

The results of the analyses of the bran proteins show that t,hese proteins contain relatively high percentages of the basic amino acids. These percentages, expressed in terms of the ash- and moisture-free proteins, are summarized in Table I. The quantity of basic amino acids in the globulin, particularly arginine and lysine, exceeds that of most other vegetable proteins which have been studied. The high percentages of tryptophane and cystine found in the albumin are also of further particular inter- est. The relatively large quantities of the so called nutritionally essential amino acids, such as lysine, tryptophane, and cystine, found in the wheat bran proteins are especially significant, from the st,andpoint of their nutritive value. Feeding experiments with rats are in progress to supplement these chemical data regarding the nutritive value of the bran proteins, and to ascer- tain the availnbi1it.y to the animals of the amino acids present.

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D. B. Jones and C. E. F. GersdorfT 243

Comparison of the Bran Proteins with the Corresponding Proteins of the Endosperm and Embryo.

Whether we look upon bran as a valuable source of protein which should be included in wheat products used for human con- sumption, or as more suitable to be used as a protein concentrate for the feeding of domest,ic animals, it is of importance to know how the bran proteins compare with thoseof the other parts of the seed, and to ascertain their amino acid composition, particularly those amino acids which have been shown to be essential for the normal nutrition of animals.

TABLE I.

Some Amino Acids oj the Proteins of Wheat Bran* (Expressed as Percentage of the Ash- and Moisture-Free Proteins.

Amino acids.

Cystine. . . . . .

Arginine. . Histidine.. Lysine. Tyrosinet.. Tryptophane$.

Prolamin. Globulin. Albumin.

per cent per cent per cent

1.65 1.38 2.80 2.29f 1.52t 3.29t 4.41 14.13 10.04 0.84 2.76 2.57 2.45 11.84 4.51 3.38 3.69 4.20 1.37 2.85 4.76

- * The percentages of the amino acids given in Table I, with the excep-

tions noted, were calculated from the results obtained by the VanSlyke method of analysis.

t Determined by the calorimetric methods of Folin and Looney (8). $ Determined by the calorimetric method of May and Rose, with modi-

fications (10).

For the purpose of comparison the figures expressing the ele- mentary composition, distribution of nitrogen, percentages of amino acids, and the free amino nitrogen of the bran proteins and of the corresponding proteins of the wheat endosperm and embryo have been assembled in Table II. The data given for t,he proteins of the endosperm and embryo are mostly figures which have been published by Osborne and associates. All of the percentages, however, given in the table for tryptophane, cystine, free amino nitrogen, and tyrosine, with the exception of the tyrosine figure for leucosin, were determined by the writ,ers.

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Proteins of Wheat Bran. II

Pro&min.--A notable difference between the alcohol-soluble protein of the bran and that of the endosperm (gliadin) is shown by their element,ary composition, part,icularly with reference to carbon and nitrogen. That the relatively low percentage of nitrogen in the bran prolamin is characteristic of this protein is indicated by the concordant results obtained by nitrogen deter- minations made on five different samples of the prolamin prepared by different methods and under varying conditions.’

The bran prolamin was found to contain about twice as much basic nitrogen as that recorded for gliadin, although its total nitrogen content was about 2 per cent lower. This difference in the nitrogen distribution of the two prolamins is to be accounted for by the higher percenOages of the basic amino acids found in the bran prolamin as compared with those of gliadin. Twice as much lysine was found in the bran prolamin as that recorded by Osborne, Van Slyke, Leavenworth, and Vinograd (2) for gliadin, and also a much higher content of arginine. The hist.idine content of the bran prolamin, on the other hand, was found to be much lower than that given for gliadin.

The estimation of trypt,ophane, tyrosine, and cystine in the bran prolamin, and in a sample of wheat gliadin especially prepared by the writers, gave closely agreeing results.

The free amino nitrogen of the bran prolamin was found to be practically three times that of gliadin.

Albumin.-About 2.9 per cent of wheat bran consists of an albumin which separates as a flocculent coagulum when its aqueous (slightly acidified with acetic acid) solution is slowly heated to about 60°C. When heated in 4 per cent sodium chlor- ide solution it coagulates at about 65°C. The albumin can be obtained nearly white when dried in the usual way, and is free from phosphorus.

Osborne (3), working wit’h commercial wheat embryo-a mix- ture consisting largely of embryo and containing more or less of the endosperm and fine bran-isolated somewhat more than 10

1 A description of the preparation and composition of these sample0 is given in a previous publication (1). The figures showing the elementary composition of the bran prolamin given in Table II are averages obtained by duplicate analyses made on the five preparations.

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D. B. Jones and C. E. I?. Gersdorff

per cent of an albumin (leucosin), which was found to coagulate at 5560°C.

Aside from the difference in their coagulation points, the two albumins also differ with respect to their composit,ion as

TABLE 11.

Analytical Data Shown for Compnrison of the Prolamin, Albumin, and Globzllin of Wheat Bran with the Corresponding Proteins of Other Parts

of the Wheat Kernel.*

(The values are expressed as percentages of the proteins).

Carbon ................... Hydrogen. ................ Sitrogen .................. Sulfur .....................

Amide N .................. Humin N ................. Basic S ................... Non-basic N ..............

Qstine ................... z I rginme ..................

Histidine .................. Lysine. ................... Tryptophane .............. Tyrosine. .................

E’ree amino ?it ............

I -- 1

Bran Pr*

lamin. ( 2lisdin. a

,er cent per cent per cent

54.25 52.72 53 21 6.75 6.86 6.71

15 35 17.66 15.42 1 35 1.14 1.35

3.58 5.12 1.20 0.18 0.07 0.52 2.30 1.00 5.12 9 53 12.25 8.92

2 29 1.76 3.29 4 41 2.97 10.04 0.84 2 19 2.57 2 45 1.21 4.51 1.37 1.09 4.76 3.38 3.30 4.20

1.88 0.65 2.84 -

oer cent per Cfni

53.02 53.43 6.84 7.40

16.80 17.76 1.28 0.91

1.16 1.06 0.43 0.29 3.50 7.73

11.83 8.82

5.94 2 83 2.75

3.34

1.52 14.13 2.76

11.84 2.85 3.69

6.59

- I

‘. 6

! ;

-

hbryo lobulin.

per cent

51.03 6.85

18.39 0.69

1.42 0.28 6 83 9.82

* The figures given for gliadin, leucosin, and the globulin of the embryo are those obtained by Osborne and associates (2, 5), with the following ex- ceptions, which were determined by the writers: All of the percentages given for cystine, tryptophane, and free amino nitrogen, and the tyrosine figure for gliadin.

t Expressed as percentage of the total nitrogen.

is shown by the data in Table II. A marked diffcrcnce is shown by their nitrogen content. Again, as in the case of the prolamins, the bran albumin, although containing decidedly less total nitro- gen than does leucosin, contains a much higher percentage of

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Proteins of Wheat Bran. II

basic nitrogen. The percentages of carbon, hydrogen, and sulfur in the two albumins, however, are in close agreement.

The percentages of the basic amino acids in the bran albumin were calculated from the results obtained by the Van Slyke method of analysis, while those given for leucosin as found by Osborne and Clapp (4) were determined by isolation according to the method of Kossel and Kutcher. Consequently we do not have a satisfactory basis upon which to make a fair comparison of the proportions of the basic amino acids in the two albumins, since the values obtained by the Van Slyke method are maximal and those obtained by Kossel and Kutcher’s method are minimal. However, the percentages found for arginine by the two methods are usually in fairly close agreement. The great difference in the percentage of arginine found in the bran albumin as compared with that given for leucosin in Table II further strongly indicates that we are dealing here with two different proteins.

The percentage of tyrosine found in the bran albumin (4.2 per cent) was determined by means of Folin and Looney’s colori- metric method. Here again a satisfactory basis of comparison is lacking, because the figure given by Osborne and Clapp (4) for tyrosine in leucosin (3.34 per cent) was based on the weight of the pure isolated amino acid. The difficulty involved in quanti- tatively separating tyrosine from mixtures of other amino acids is well known. It is therefore probable that no significant differ- ence esists in the percentages of tyrosine in the two albumins.

Globulin .-Osborne and Voorhees (5) obtained from a “straight” wheat flour about 0.6 per cent of a globulin. Later, Osborne and Campbell (6) found that about 5 per cent of commercial wheat embryo consists of globulin. They found no difference in the com- position of the globulins obtained from these two sources.

The bran globulin is precipitated from a 4 per cent sodium chlor- ide solution by the addition of ammonium sulfate until 0.4 to 0.65 of saturation is reached. It coagulates in a slightly acidified 4 per cent sodium chloride solution at 95°C. Regarding the globulin of the embryo, Osborne states (3) that it is precipitated by saturating its solutions with magnesium sulfate, but not with sodium chloride. Dissolved in 10 per cent sodium chloride solu- tion, it is partly but not completely coagulated at temperatures below 1OO’C.

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D. B. Jones and C. E. F. Gersdorff 247

A marked difference between the bran globulin and the globulin isolated by Osborne and Campbell is indicated by their elementary composition as shown in Table II. The bran globulin is also higher in basic nitrogen.

Especially noteworthy in connection with the bran globulin are the high percentages of arginine and lysine (Tables I and V). No data showing the amino acid composition of the embryo globu- lin are available for comparison. The bran globulin was found to be free from phosphorus.

From the comparative data which have been presented it is clear that there can be but little doubt, if any, that the proteins obtained from wheat bran are distinctly different from the cor- responding proteins of the embryo and endosperm Further studies of the bran proteins are in progress.

EXPERIMENTAL.

The bran proteins used for the analyses herein described were prepared from bran which originally came mostly from 75 per cent Dark 1 Northern Spring Wheat and 25 per cent of 2 Hard Kansas Wheat. A detailed description of how the bran was prepared and of the separation and elementary composition of the proteins has been given in a previous publication (1).

Analyses of the Proteins by the Van Xlyke.Method.

Duplicate samples of 3 gm. each of the bran proteins were hydro- lyzed by boiling for, about 30 hours with 100 cc. of 20 per cent hydrochloric acid. The phosphotungstates of the bases were decomposed in the usual way with a mixture of ether and amyl alcohol. The results of the analyses are given in Tables I, III, IV, V, and VI.

The figures expressing the percentages of the basic amino acids in the proteins, as calculated from the results obtained by the Van Slyke analyses, are included in Table I.

The distribut,ion of nitrogen in the proteins as calculated in terms of percentage of the proteins is given in Table VI.

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Proteins of Wheat Bran. II

TABLE III.

Distribution of Nitrogen in the Wheat Bran Prolamin as Determined by the Van Slyke Method.*

Sample I, ash-andmoisture-free, 2.7390gm. protein, 0.4262gm.nitrogen.t

“ II, “ ‘I “ 2.7390 “ “ 0.4262 “ “ t

I II I II Aver- age.

______ ______ ImL. urn. per cent per cent per cent

Amide N . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.09820.0978 23.04 22.95 23.06 Humin N adsorbed by lime.. . . . . . 0.0042 0.0038 0.99 0.89 0.94

“ “in ether-amyl alcohol extract. 0.00090.0012 0.21 0.28 0.24 Cystine N.............................. 0.00510.0054 1.20 1.27 1.23 Arginine N.............................0.03870.0390 9.08 9.15 9.12 Histidine N. . . . . . . . . . . . . . 0.0054 0.0070 1.27 1.64 1.45 Lysine N . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.01360.0121 3.19 2.84 3.02 Amino N of filtrate.. . . 0.2399 0.2385 56.29 55.96 56.13 Non-amino N of filtrate. . . 0.0224 0.0210 5.26 4.93 5.09

-_________

TotalN regained.....................0.42840.4258100.53 99.91100.22

* Nitrogen figures corrected for the solubilities of the phosphotung- states of the bases.

t Nitrogen content of the protein, 15.56 per cent.

TABLE IV.

Distribution of Nitrogen in the Wheat Bran Albumin as Determined by the Van Slylce Method.*

Sample I, ash- and moisture-free, 2.7225 gm. protein, 0.4250gm. nitr0gen.t “

II, “ (‘ ‘( 2.7225 ” Ii 0.4250 “ “ t -

II I II AlW-

L------ age.

gm. *In. pw cent per cent per cent Amide N _..._........_._............... 0.03250.0326 7.65 7.67 7.66 Humin N adsorbed by lime.. . . . 0.0130 0.0133 3.06 3.13 3.09

“ “in ether-amyl alcohol extract. 0.00100.0009 0.24 0.21 0.22 Cystine N ._........,...,.....,......... 0.00870.0091 2.05 2.14 2.10 Arginine N _... ._...,....___... ~ _.___._. 0.08810.0878 20.73 20.66 20.70 Histidine N............................0.01960.0182 4.61 4.28 4.45 Lysine N . . . .._..__..._..._............. 0.02360.0235 5.55 5.53 5.54 Amino N of filtrate. _. _. . . . . . O-23880.2396 56.19 56.38 56.28 Non-amino N of filtrate.. . 0.00310.0037 0.73 0.87 0.80

~__~~___

TotalNregained . . . . . . . .._..... 0.42840.4287100.81100.87100.84

* Nitrogen figures corrected for the solubilities of the phosphotungstates of the bases.

t Nitrogen content of the protein, 15.61 per cent.

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D. B. Jones and C. E. F. Gersdorff 249

TABLE V.

Distribution of Nitrogen in the Wheat Bran Globulin as Determined by the Van Slyke Method.*

Sample I, ash- andmoisture-free, 2.8344 gm. protein, 0.5105 gm. nitr0gen.t “ 11 “ L‘ “

> 2.8344 “ “ 0.5105 “ (‘ t

I II I II Aver- age.

~__

Urn. Pm. per cent per cent per cent

Amide N... ._.,.,......._._...,._...__. 0.03010.0300 5.90 5.87 5.89 Humin N adsorbed by lime.. . 0.0065 0.0070 1.27 1.37 1.32

‘C “ in ether-smyl alcohol extract. 0 0013 0 0015 0.25 0.29 0 27 Cystine N .______...........,..,.,.___,. 0.00460.0045 0.90 0.88 0.89 Arginine N............................. 0.12890.1289 25.25 25.25 25.25 Histidine N.. _. _. _, 0.02090.0215 4.09 4.21 4 15 Lysine PI‘.. . . 0.06420.0644 12 58 12.62 12.60 Amino N of filtrate.. . 0.2358 0 2353 46.19 46 09 46.14 Non-amino N of filtrate. 0.0146 0.0144 2.86 2.83 2 84

~-~

TotalN regained..................... 0.50690 5075 99.29 99.41 99 35

* Nitrogen figures corrected for solubilities of the phosphotungstates of the bases.

t Nitrogen content of the protein, 18.01 per cent.

TABLE \-I.

Distribution of Nitrogen in the Wheat Bran Proteins as Determined from the

Van. Slyke Analyses in Terms of Percendagw of the Proteins.

Amide ............................. IIumin ............................ &sic .............................. Non-basic .........................

Nitrogen. Prolamin.* Globu1in.t I Albumin.: I

per cell t per cent per cent

3.58 1.06 1.20 0.18 0.29 0.52 2 30 7.73 5.12 9.53 8.82 8.92

-

15.76 Total.. . . . 15.59 1 17.90

* Sitrogen content 15.56 per cent. t Nitrogen content 18.01 per cent. 1 Nitrogen content 15.61 per cent.

Free Amino Nitrogen.

Quantities equivalent to 0.75 gm. of the moisture-free bran proteins and also of gliadin were dissolved in 2.5 cc. of glacial

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250 Proteins of Wheat Bran. II

acetic acid plus 1 cc. of distilled water, and the solutions diluted with water to 100 cc. 2 cc. of each of the protein solutions in duplicates were used for the determination of t,otal nitrogen and amino nitrogen. Caprylic alcohol was used t.o prevent foaming, and the larger reaction bulb of t.he Van Slyke apparatus was used in combination with the micro gas burette. The results are given in Table VII.

It has been shown by Van Slyke and Birchard (7), and others, that the free amino nitrogen of many proteins corresponds closely to one-half of the lysine nitrogen. This relationship was found to hold true both in the case of the bran proteins and in that of

TABLE VII.

Free Amino Nitrogen of the Wheat Bran Proteins and of Wheat Gliadin.

Protein.

Prolamin (bran). ...... Albumin ( “ ) ....... Globulin ( “ ). ...... Glindin (wheat). ......

:: 3 0 01 c 2 G s -2 2

3 a __-

mg. cc. 9.48 0.33

10.01 0.53 7.23 0.88 7.44 0.09

Fi d 2 5 b I cc

3 2 __-

mm. “C.

762 30 762 31 762.1 30 762 30

T 0 ;

6 ,” .- 2 a E G ;zi s 23 .- 6 12 p: A- mv. per cent per cent

0.1786 1.88 1.51 0.2841 2.84 2.77 0.4765 6.59 6.30 0.0487 0.65 0.66*

* Calculated from the results obtained by Osborne and associates.

gliadin. It is noted that the ratio of free amino nitrogen to total nitrogen in the case of the bran prolamin is nearly three t,imes that found in gliadin.

Cystine, Tyrosine, and Tryptophane Estimations.

The cystine, tyrosine, and tryptophane contents of the bran proteins, and also of gliadin, were estimated calorimetrically. Folin and Looney’s method (8) was used for cystine and tyrosine, and that of May and Rose (9) for tryptophane. A detailed account of these estimations h&s been given in a recent publica- tion (10). These results are here incorporated in Tables I and II.

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

SUMMARY.

The proteins of wheat bran, consisting essentially of a prolamin, a globulin, and an albumin, have now been analyzed by the Van Slyke method, and the distribution of nitrogen and the percentages of the basic amino acids have been estimated. The percentages of tyrosine, tryptophane, and cystine have also been estimated by calorimetric methods. The wheat bran proteins, in general, are characterized by a high content of the basic amino acids. This is particularly true of the albumin and globulin. The quantity of basic amino acids found in the globulin exceeds that of most other vegetable proteins which have been studied. The following per- centages of amino acids were found: (1) In the prolamin; cystine 2.29, arginine 4.41, histidine 0.84, lysine 2.45, tryptophane 1.37, and tyrosine 3.38; (2) in the albumin; cystine 3.29, arginine 10.04, histidine 2.57, lysine 4.51, tryptophane 4.76, and tyrosine 4.20; and (3) in the globulin; cystine 1.52, arginine 14.13, histidine 2.76, lysine 11.84, tryptophane 2.85, and tyrosine 3.69.

Marked differences were found between the bran proteins and the corresponding proteins of the wheat embryo and endosperm, both with regard to their elementary composition, distribution of nitrogen, and amino acid content.

The significance of the high percentages of the so called nutri- tionally essential amino acids found is pointed out with respect to the nutritive value of the bran proteins.

BIBLIOGRAPHY.

1. Jones, D. B., and Gersdorff, C. E. F., J. Biol. Che~n., 1923-24, lviii, 117. 2. Osborne, T. B., Van Slyke, D. D., Leavenworth, C. S., and Vinograd,

M., J. Biol. Chem., 1915, xxii, 259. 3. Osborne, T. B., Carnegie Institution of Washington, Pub. No. S:, 1907,

113. 4. Osborne, T. B., and Clapp, S. H., Am. J. Physiol., 1906-07, xvii, 231. 5. Osborne, T. B., and Voorhees, C. G., Am. Chem. J., 1893, xv, 392. 6. Osborne, T. B., and Campbell, G. T., J. Am. Chem. Sot., 1900, xxii, 379. 7. Van Slyke, D. D., and Birchard, F. J., J. Biol. Chem., 1913-14, xvi, 539. 8. Folin, O., and Looney, J. M., J. Biol. Chem., 1922, li, 421. 9. May, C. E., andRose, E. R., J. Biol. Chem., 1922, liv.‘213.

10. Jones, D. B., Gersdorff, C. E. F., andMoeller, n., J. Biol. Chem., 1924- 25, Ixii, 183.

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D. Breese Jones and C. E. F. GersdorffWHEAT ENDOSPERM AND EMBRYO

CORRESPONDING PROTEINS OFPROTEINS WITH THE

COMPARISON OF THE BRANOF FREE AMINO NITROGEN: A

PERCENTAGES OF AMINO ACIDS ANDDISTRIBUTION OF NITROGEN,

PROTEINS OF WHEAT BRAN: II.

1925, 64:241-251.J. Biol. Chem. 

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