THE NATURE AND IDENTITY OF WHEAT GLUTENIN.”

BY M. J. BLISH AND R. M. SANDSTEDT.

(From the Agricultural Experiment Station, University of Nebraska, Lincoln.)

(Received for publication, September 14, 1929.)

INTRODUCTION.

The gluten of wheat flour has always been of outstanding importance and interest to the wheat industry in general and to the cereal chemist in particular. Osborne’s (1907) well founded conclusion that gluten consists primarily of two distinct and in- dividual proteins, glutenin and gliadin, and his characterization of these two proteins has for many years served as the foundation for most modern thought as to their nature and respective in- dividualities.

Of these two proteins glutenin has within recent years come into special prominence, due largely to evidence obtained by Sharp and Gortner (1923) indicating that glutenin is the protein that is solely responsible for variations among the colloidal properties of glutens from different flours, and that these variations may in turn be important causes of differences among the bread-making characteristics of the flours in question.

Both proteins have been frequently isolated and purified by various investigators, and their constitution and properties have been studied by such methods as have been available. The nitro- gen distribution method of Van Slyke has been chiefly relied upon to indicate chemical constitution. There is ample justihca- tion for the belief that the values yielded by Van Slyke’s pro- cedure are, for the most part, far from being absolute, even though they may be highly informative. Results of protein analyses by this procedure are at best strictly comparable only when the same manipulative conditions are maintained in all instances.

* Published with the permission of the Director as Paper No. 82, Journal Series, Nebraska Agricultural Experiment Station, Lincoln.

195

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196 Wheat Glutenin

Various workers have had occasion to prepare the two gluten proteins, glutenin and gliadin, in as pure form as possible, and have subjected their preparations to Van Slyke analysis. In almost all instances the methods of preparation and purification of these proteins have either followed closely or have been based upon the methods originally used and described by Osborne. On comparison of different analyses of the same protein, it is at once noticeable that there is greater concordance among the results of analyses of gliadin than is correspondingly true with glutenin. One cannot escape the conviction that the chemical identity of the former is far more definitely established than that of the latter.

TABLE I.

Values for Amide Nitrogen and jor Arginine Nitrogen in Various Gliadin Preparations.

Analyst.

Osborne (1924).. . Van Slyke (1911).. . . . . . Csborne, et al. (1915). . . Blish (1916). . .

,‘ “

“ and Sanhbtkbt’ (1924) .‘_‘. 1: : : : : Cross and Swain (1924). , .

‘L ‘L ‘L ‘I . . . . . . . . . . “ “ ‘I “ . . ‘< L‘ I‘ “ . . . . . . . .

Hoffman and Gortner (1927). . . . . Dill and Alsberg (1925). .

-

- _ Source of flour. hide N.

Unknown. I‘ ‘I

Spring wheat. Soft “ Hard winter. Idaho. Patent. Club. Fortyfold.

per cent 24.5 25.5 24.6 26.1 25.9 26.4 26.8 26.4 26.4 26.2 25.9 26.2

ArgiPfine

per cent

5.71 5.45 4.55 4.47

4.86 5.06 5.21 4.78 5.29

In considering variations among results of Van Slyke analyses of different preparations of the same protein, attention may ap- propriately be confined to the values for amide nitrogen and for arginine nitrogen. These two constituents are determined simply and directly, and their estimation may be regarded as subject to less error than is involved in the estimation of the other Van Slyke units. Comparative data of this nature for gliadin and glutenin are shown in Tables I and II, re&ectively.

Inspection of data in Tables I and II discloses far greater vari- ability among analyses for glutenin than for gliadin. In the case

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M. J. Blish and R. M. Sandstedt 197

of gliadin, all values for amide nitrogen fall within extremes of 24.5 to 26.8 per cent, with 7 of the 12 values falling close to 26. The extremes for arginine nitrogen in gliadin are 4.47 and 6.38 per cent, with most values reasonably concordant. Among the glu- tenin samples, on the other hand, the range of values for amide nitrogen is 12.4 to 18.8 per cent, and for arginine nitrogen the range is 8.18 to 12.94 per cent.

The situation becomes all the more conspicuous when the comparison of variability among gliadin preparations with that of glutenin preparations is confined to samples all of which were prepared in the same laboratory, opportunity for such a compari-

TABLE II.

Values for Amide Nitrogen and for Arginine Nitrogen in Various Glutenin Preparations.

Analyst. Source of flour. I I

Amide N . Argi?e

Osborne (1924).. . . . . . Blish (1916). .

<‘ “ . . . . . . . . . . Cross and Swain (1924). . .

“ “ ‘I “ . ‘< “ I‘ “ . . . . ,I ‘I “ ‘I

Hoffman and Gortner (1927): .‘,‘.’ Larmour (1927).. . : Blish (unpublished).. .

L‘ I‘

Unknown. Spring wheat. Soft “ Idaho. Patent. Club. Fortyfold. Patent.

“

Hard winter. “ “

per cent

18.8 16.5 16.2 15.6 16.0 14.2 13.1 13.6 14.8 13.2 12.4

per cent

9.69 9.27

10.10 8.18 9.23

12.94 11.96 10.90 12.50 11.20

son being afforded by the data of Cross and Swain (1924). They selected four different types of wheat and made carefully prepared preparations of gliadin and glutenin, respectively, from each lot. Their extreme values for amide nitrogen among the four samples of gliadin were 26.2 and 26.8, and values for arginine nitrogen fell between the limits 4.78 and 5.21. Among the four glutenin samples, on the other hand, values for amide nitrogen range from 13.1 to 15.6, and those for arginine nitrogen run from 8.18 to 12.94, the latter values incidentally being not only the extremes for the one laboratory, but for all workers whose data appear in Table II.

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198 Wheat Glutenin

Cross and Swain (1924) regard their data as evidence that glia- dins from different wheats are identical, and such a conclusion is obviously justified. However, they conclude that glutenins from different wheats are also identical, a decision for which there is far less justification. The data for glutenin would seem rather to indicate that one of two things is true: (1) either glutenins from different wheats are not identical, or (2) methods for the prepara- tion and purification of glutenin are uncertain and unreliable, and the true chemical nature, identity, and individuality of this protein have not been satisfactorily established. This communi- cation is a preliminary report of certain experiments bearing upon the latter possibility.

Glutenin, according to Osborne’s (1924) characterization, is that portion of the wheat flour protein that is left after oomplete removal of all protein material soluble in dilute neutral salt solu- tions and in 50 to 70 per cent alcohol. It is readily soluble in dilute alkali, and this reagent has invariably been used as the initial solvent or dispersing agent in the isolation of the glutenin, which is later precipitated from the filtered extract byneutraliza- tion with acid. That treating protein with alkali tends to produce certain alterations in the protein molecule is well known. Among the effects known to be possible are liberation of ammonia, de- struction of cystine, splitting of arginine into ornithine and urea, and racemization. The rate at which these alterations occur depends of course upon factors such as concentration of OH ions, temperature, and time of exposure. In the preparation of the cereal glutelins, however, the alkali has been of such dilution, and other factors have been such that serious alteration of the protein molecule has not generally been suspected. That this possibility should be taken more seriously becomes evident when Hoffman (1925) reports that cystine can no longer be isolated from human hair after it has received such a mild treatment as washing with hot 1 per cent sodium carbonate solution. In the present studies one of the first items to be considered has been the effect of different concentrations of alkali used in extracting the glutenin, upon the nitrogen distribution in the resulting product after its complete hydrolysis in strong acid.

In the first series of experiments herewith reported, glutenin preparations were isolated from two different lots of the same

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M. J. Blish and R. M. Sandstedt 199

flour, the flour having previously been extracted with 5 per cent K&SO4 solution (followed by distilled water) to remove albumin and globulin, and dried in vacua at a low temperature. A 40 gm. portion of this flour was extracted for 20 minutes, with constant shaking, with 300 cc. of lv/60 NaOH. An equal portion was simi- larly extracted with 300 cc. of 0.2 N NaOH. Each lot was then made up to 1 liter with 95 per cent methyl alcohol, according to the procedure recommended by Blish and Sandstedt (1924). This gives a final alcoholic concentration from which the starch rapidly settles, permitting ease of filtration, and the alcohol also retains the gliadin in solution when the extract is neutralized in order to precipitate the glutenin. After filtration, a suitable por- tion of each extract was neutralized with HCI to the point of optimum flocculation of the glutenin. After flocculation and

TABLE III.

Total, Amide, and Basic Nitrogen in Glutenins A and B, after Complete Hydrolysis.

Strength of alkali

Preparation. used in initial Total N in extraction of hydrolysate. Amide N. Basic N.

glut&n.

gm. per cent pm cent

A x/60 0.1393 21.4 9.70 B N/5 0.1093 18.1 13.1

settling, the clear supernatant liquids were poured off, and the precipitates were washed repeatedly by decantation with 65 per cent methyl alcohol solutions containing respectively the cal- culated concentrations of NaCl as formed by neutralization of the original extracts. Each preparation was then completely hydrolyzed with strong HCl, and Hausmann numbers were de- termined, with results as presented in Table III. These glutenins as prepared by the weaker and the stronger alkali are designated, respectively, as Glutenins A and B.

Data in Table III show a marked difference in the percentages of both amide and basic nitrogen as produced by different alkali concentrations used for dispersing and extracting the glutenins originally, the higher alkali concentration causing a marked de- crease in amide nitrogen and a very pronounced increase in basic or diamino acid nitrogen.

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200 Wheat Glutenin

These experiments were repeated, the same procedure being followed in all respects, except that the glutenin was precipitated by neutralizing the alkaline extracts with COZ instead of HCl. The results are shown in Table IV.

Although the actual values in Table IV are slightly different from those in Table III, the comparative effects of the different alkali concentrations are shown to be the same as in Table III.

The next experiment involved preliminary extraction of the same flour with 0.05 N and 0.25 N NaOH solutions, respectively, followed by dilution of the extracts with methyl alcohol, as before, and precipitation of glutenin from the filtered extracts by neu- tralization with Cog. In this instance, however, instead of merely washing the precipitated glutenins by decantation, they were

TABLE IV.

Total, Amide, and Basic Nitrogen in Glutenins A and B, the Glutenins Having Been Precipitated from Their Alkaline Extracts by

Neutralization with Carbon Dioxide.

Strength of alkali Preparation. used in initial Total N in

extraction of hydrolysate. Amide N. glutenin.

gm. per cent A N/60 0.205 20.3 B N/5 0.125 16.9

-

Basic N

per cent

11.51 14.75

L

redissolved and reprecipitated several times from dilute alcoholic alkali in order to remove all possibility of adhering or occluded gliadin. The preparation that was originally dispersed by 0.05 N

alkali showed, after hydrolysis, an amide nitrogen content of 15.9 per cent, whereas a value of only 12.1 per cent was found in the case where 0.25 N alkali had been the preliminary dispersing agent. These values are of a sufficiently lower order than those secured in the preceding experiments to indicate that the several redispersions and reprecipitations used for purification purposes caused a sub- stantial lowering of both values for amide nitrogen, comparative values remaining in the same order, however. The values for basic nitrogen were more than twice as high as in the preceding experiments, being 22.1 per cent for the preparation involving the weaker NaOH, and 24.5 per cent for the stronger. The same

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M. J. Blish and R. M. Sandstedt

tendency persists as to correlation between strength of alkali and per cent of basic nitrogen. These results are typical of many ex- periments subsequently performed and involving concentration of NaOH as the sole variable.

In view of this situation it was considered desirable to attempt the preparation of glutenin under conditions avoiding exposure to alkaline reaction at any stage of the procedure. At first, numerous attempts based upon acid dispersion failed because of inherent difficulties in physical manipulation. Glutenin appar- ently is dispersed in very dilute acid with difficulty and only under certain conditions. When thus dispersed, its ultimate particles are so large, due either to large aggregates or to high degree of hydration, or both, that it will not pass through a filter to permit elimination of starch and other solid foreign material. Further- more it was difficult to find a method for coagulating it from acid dispersion without also precipitating gliadin in physical combina- tion. Definite and uniform preparations were, however, finally secured by the following procedure: Crude gluten is prepared from flour by kneading the flour-water dough under tap water in the usual manner. This gluten is finely macerated and placed in a large volume of very dilute acetic acid. After standing over- night it is thoroughly dispersed, giving a somewhat viscous solu- tion with starch and other solid foreign material in suspension. Filtration being impossible, the solution is diluted with methyl alcohol until an alcoholic concentration of 65 to 70 per cent is obtained. The dilute alcoholic solution is then passed slowly through a Sharples supercentrifuge, whereby starch and other suspended matter are removed, giving a highly opalescent sol. When this solution is neutralized to a pH slightly below 7, by slowly stirring in N NaOH solution (but never allowing the material to become alkaline), a heavy, gelatinous protein precipitate comes down rapidly. The gliadin remains in the alcoholic solution, and is at once decanted off. The precipitate may be redispersed in dilute acetic acid, and reprecipitated after the solution is again diluted with methyl alcohol. After two or three such treatments, followed each time by washing with water, the gliadin is completely eliminated as shown by absence of protein in the supernatant liquid. The precipitated glutenin is then dried in the usual manner with alcohol and ether. It was noted that a satisfactory

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202 Wheat Glutenin

separation of glutenin could not be obtained when ethyl alcohol was substituted for methyl alcohol. The reason for this is doubt- less associated with the known greater dehydrating power of methyl alcohol.

Preliminary observations and studies of several samples of glutenin prepared in this manner indicate that it differs both physically and chemically from products whose preparation has involved more or less prolonged exposure to alkaline reaction at intervals during the process. There are both physical and chemi- cal differences. When freshly precipitated, the new protein is far more coherent and gelatinous than are preparations precipi- tated from alkaline extracts, the latter being decidedly flocculent and non-coherent. The new protein bears a close resemblance physically to the original crude gluten, lacking only in elasticity and toughness.

The nitrogen content is probably close to 17.50 per cent. One sample showed 17.4 per cent nitrogen, as corrected to a moisture- and ash-free basis. It is difficult completely to eliminate the starch, and all samples thus far have shown traces of starch or dextrin to the extent that they do not give a water-clear solution in alkali until this minute quantity of foreign matter has settled out. The amide nitrogen after acid hydrolysis has in all cases run close to 22 per cent of the total nitrogen, this value being decidedly higher than any recorded for glutenin preparations involving alkali (see Table II), and being only about 4 per cent below the average of recorded values for gliadin (see Table I). Percentages of arginine nitrogen were estimated in these prepara- tions by the procedure recently suggested by Plimmer (1925) ; that is to say, the entire filtrate from the amide nitrogen deter- mination was used for prolonged boiling with strong NaOH, instead of first precipitating the diamino acids with phosphotung- stic acid as in the customary procedure. This method used gives somewhat higher results than the usual procedure, since it prob- ably accounts for some arginine that would escape precipitation by phosphotungstic acid. The arginine values for all preparations of the new glutenin ran close to 9 per cent of the total nitrogen. This indicates a lower percentage of arginine in the new than in the old preparations.

That the new preparation is in no way physically contaminated

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M. J. Blish and R. M. Sandstedt 203

with gliadin is evidenced by the fact that the dry and finely pow- dered material yielded no trace of protein upon prolonged extrac- tion with 70 per cent alcohol.

Preparations of glutenin resembling those made according to the conventional method (see Table II) may readily be produced from the new protein by dissolving it in alkali, and later precipitat- ing by neutralizing the alkaline solution. Thus three samples of the new preparation were dispersed in solutions of 0.02 N, 0.1 N,

and 0.2 N NaOH, respectively. After standing overnight the solutions were diluted with alcohol and were neutralized by treat- ment with COZ. The precipitates were washed, hydrolyzed, and tested for amide nitrogen, yielding 16.85 per cent, 12.53 per cent, and 10.32 per cent respectively, the per cent of amide nitrogen being as usual inversely proportional to the concentration of alkali.

The ultimate particles, or molecules, as the case may be, of the new protein are apparently of greater size and complexity than those of glutenin as ordinarily prepared by methods involving extraction with and exposure to alkali. This was indicated by examining the protein remaining in solution after precipitation of the glutenin by neutralization of the alkaline solutions in the immediately preceding experiments. The chemical constitution of this protein remaining in the supernatant liquid differs from that of glutenin prepared either by the alkali or acid dispersion method. Upon hydrolysis it yields approximately 25 per cent of its total nitrogen as amide nitrogen, and its percentage of arginine nitrogen runs about 7 to 8 per cent. These values are close to the figures obtained with pure gliadin. Whether or not it actually is identical with gliadin is now under investigation. The fact that it cannot be removed by alcohol alone, but is split off only after treatment with alkali strongly suggests that the protein that has customarily been regarded as glutenin does not exist as such in the original flour, but is rather a product of the action of alkali upon a larger and more complex protein body.

When glutenin is precipitated from alkaline alcoholic extracts of flour or gluten, not only does the composition of the precipitate vary with the strength of the alkali, but the amount of protein precipitated varies inversely as the strength of the alkali, as has been shown by Blish (1926). Thus the quantity of protein re-

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204 Wheat Glutenin

maining in the supernatant liquid will vary also. It is of interest, however, that experimental evidence thus far obtained in this laboratory indicates that although the precipitated glutenin varies both in amount and composition, under these conditions, the pro- tein left in the supernatant liquid varies in amount only, and not in composition. This adds support to the idea that the gluten of wheat flour contains, in addition to gliadin, a protein body that is more complex than either gliadin or what is generally regarded as glutenin. The effect of alkali upon this larger protein entity is to split off protein resembling gliadin in properties thus far studied, the amount split off depending upon the strength of the alkali, or conditions of exposure thereto. The remaining fraction represents glutenin as it has ordinarily been prepared and studied, and the nature of the alkali treatment will largely determine both its quantity and its chemical constitution.

In view of the inherent difficulties involved in definitely estab- lishing the identity or individuality of proteins, and since the work thus far completed is preliminary in character, it would be hazard- ous at this stage to attempt to classify the new preparation as a distinct and individual protein. Some nine or ten samples have been prepared. They agree within a reasonable factor of error both as to per cent of amide nitrogen (averaging about 22 per cent) and arginine nitrogen (averaging about 9 per cent). This indi- cates a definite and consistently uniform composition.

It is of course improbable that any protein may be isolated from biological material and subjected to the customary methods of purification without undergoing either physical or chemical al- teration, or both. As to the new glutenin preparations herewith described, there is substantial assurance that far less alteration has occurred than is ordinarily the case with preparations made according to the principles usually and heretofore employed.

Whatever may be the nature or importance of the new “glu- tenin,” these experiments clearly indicate an irreversible altera- tion in the chemical structure of a considerable portion of flour or gluten protein when dispersed in an alkaline medium, regardless of alkali concentration. It is likely that this factor has also in- fluenced the composition and properties of glutelins that have been prepared from other cereals as well. Kondo and Hayashi (1926) after experimenting with rice glutelin have recently stated

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M. J. Blish and R. M. Sandstedt 205

their conviction that it is not possible to prepare individual pro- teins whose chemical identities are established beyond all doubt, especially where temporary solution in alkali is involved. They find that rice glutelin is very susceptible to alteration even when the alkali is very dilute, and express the belief that the best that may be expected under such circumstances is a protein that is well defined and reproducible. Their experiences, together with those of the present writers, serve as a substantial confirmation of the statement that appears in Osborne’s monograph on “The Vegetable Proteins” (1924 edition) when he said in speaking of the glutelins: “Very little that is definite has been learned respecting these proteins and until they have been further studied by modern methods it is hopeless to discuss them further.”

SUMMARY.

1. Glutenin, as prepared by customary methods involving ex- traction with or temporary solution in alkali, is a product result- ing from an irreversible alteration by the action of alkali on a more complex protein body.

2. Both yield and chemical constitution of glutenin prepared by the usual methods will vary with the concentration of alkali.

3. A new “glutenin” has been prepared by a procedure in which exposure to alkali is avoided at all stages. It differs from the usual glutenin both in physical properties and in chemical constitution.

4. It is probable that some irreversible alteration occurs when any protein material is dispersed in alkaline solution, regardless of the concentration of alkali.

5. There is occasion for further intensive investigation of the nature of the nitrogenous material of wheat and flour, and of the other cereals as well, and such investigation will doubtless lead to a substantial revision of present day ideas as to the true character of this protein or group of proteins, as the case may be. This situation applies with special force to the so called cereal “glutelins.”

BIBLIOGRAPHY.

Blish, M. J., J. Znd. andEng. Chem., 8, 138 (1916). Blish, M. J., J. Assn. OjWaZ Agric. Chemists, 9, 417 (1926).

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Blish, M. J., and Sandstedt, R. M., Cereal Chem., 2,57 (1924). Cross, R. J., and Swain, R. E., Ind. and Eng. Chem., 16,49 (1924). Dill, D. B., and Alsberg, C. L., .I. Biol. Chem., 65,279 (1925). Hoffman, W. F., J. Biol. Chem., 66,251 (1925). Hoffman, W. F., and Gortner, R. A., Cereal Chem., 4,221 (1927). Kondo, K., and Hayashi, T., Mem. CoZl. Agric., Kyoto Imp. Univ., 2, 37

(1926). Larmour, R. K., J. Agric. Research, 36,109l (1927). Osborne, T. B., Carnegie Institution of Washington, Pub. No. 8.$ (1907). Osborne, T. B., The vegetable proteins, 2nd edition, London (1924). Osborne, T. B., Van Slyke, D. D., Leavenworth, C. S., and Vinograd, RI.,

J. BioZ. Chem., 22,259 (1915). Plimmer, R. H. A., and Rosedale, J. L., Biochem. I., 19,102O (1925). Sharp, P. F., and Gortner, R. A., Minnesota Agric. Exp. Station Techn. Bull.

19 (1923). VanSlyke, D. D., I. Biol. Chem., lo,15 (1911-12).

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M. J. Blish and R. M. SandstedtWHEAT GLUTENIN

THE NATURE AND IDENTITY OF

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