T H E PHYSICO-CHEMICAL PROPERTIES OF STRONG AND WEAK FLOURS, V. T H E IDENTITY OF T H E GLUTEN PROTEIN RESPONSIBLE FOR T H E CHANGES I N HYDRATION CAPACITY PRODUCED BY ACIDS* BY PAUL FRANCIS SHARP AND ROSS AIKEN GORTNER**
In the preceding paper of this series’ a method was described for the reduction of the concentration of the soluble electrolytes present in the original flour down to a point where they exerted little effect on increased imbibition produced by various acids. Later it was found that the centrifuging of the flour-in-water suspension advocated in the method, could be replaced by simple decantation, the two methods giving identical results and the decantation method being much easier. I n the decantation method the weighed amount of flour was made up to 1 liter with distilled water and shaken a t intervals over a period of 45 minutes, the material was then allowed to settle for 10 to 15 minutes, the supernatant liquid decanted, the residue shaken with an additional 500 cc of distilled water, the material allowed to settle, the supernatant liquid decanted and the residue made up to a total volume of 100 cc. This 100 cc was then taken for the viscosity determination according to the method previously described. We had suspected for some time that the glutenin was * From sota.
the Division of Agricultural Biochemistry, University of Minne-
** Published with the approval of the Director as Paper No. 367, Journal Series, Minnesota Agricultural Experiment Station. Presented before the Division of Biological Chemistry of the American Chemical Society a t the Fall Meeting, Pittsburgh, Pa., Sept. 7, 1922. Abstracted from portion a of a thesis presented by Paul F. Sharp t o the Graduate School of the University of Minnesota in partial fulfillment of the Degree of Doctor of Philosophy, June 1922. 1R.A. Gortner and P. F. Sharp: “The Physico-Chemical Properties of Strong and Weak Flours, IV.” Jour. Phys. Chem., 27, 567 (1923). a R. A. Gortner and P. F. Sharp: “The Physico-Chemical Properties of Strong and Weak Flours, 111.” Ibid., 27, 481 (1923).
Physico-Chemical Propeyties of Flours.
V.
675
the protein mainly responsible for the changes in imbibition of the flour or of the gluten under the influence of acids and alkalies. The determinations reported in this paper were carried out in order t o test this supposition and to ascertain which protein and what quantity of protein was removed along with the electrolytes in the extraction method described above. Duplicate 9 gram and 18 gram portions'of flour 1009 (a strong patent flour, milled from hard red spring wheat grown in the Red River Valley) were extracted for the removal of the electrolytes by the centrifuge method and the amount of protein (N X 5.7) removed by this treatment was determined. The flour residue was then extracted for two hours in a mechanical shaker with alcohol, 100 cc of 95 percent alcohol being added to the residue which contained enough water to dilute the alcohol t o about 70 percent. The alcohol was removed by centrifuging and the flour residue was treated with two successive portions of 100 cc each oi 70 percent alcohol. The three alcoholic extracts were combined and the protein thus removed was determined. The flour residue was again treated with 50 cc of 70 percent alcohol and agitated in a mechanical shaker for 30 minutes, the mixture centrifuged, and the protein in the alcohol again determined. The residue was extracted a third time in a similar manner and the protein in the extract again determined. The residue was then extracted with 50 cc of 95 percent alcohol followed by 50 cc of ether and the protein in these extracts determined. The residue was then air dried and finally suspended in 100 cc of distilled water and the imbibition capacity of the remaining protein was measured with a MacMichael viscosimeter. The highest viscosity produced with lactic acid acting on this flour residue, and the protein (N X 5.7) removed in the various extracts are given in Table I. It is seen from Table I that the treatment with water removes a considerable proportion of the protein, and the results obtained by subsequent extraction with alcohol show that the protein removed is the alcohol soluble protein, gliadin,
Paul F . Sharp and Ross A.Gortner
676
for although t h e individual values in the t w o columns vary considerably, nevertheless the sums of the t w o show good
TABLE I Protein extracted from flour 1009 by (1) two successive extractions of 1 liter and 500 cubic centimeters of water, (2) residue extracted three successive times with 100 cc portions of 70 percent alcohol, (3) is the sum of (1) (2), (4) residue extracted with 50 cubic centimeters of 70 percent alcohol, (5) residue extracted again with 50 cubic centimeters of 70 percent alcohol, (6) residue from (5) extracted twice with 50 cubic centimeters of 93 percent alcohol followed by 50 cubic centimeters of ether, (7) sum of protein extracted, (8) dry residue was made up to 100 cubic centimeters with water and the highest viscosity obtained with lactic acid determined. The torsion wire read 29.2" with GOYo sucrose solution (viscosity 43.SG centipoise).
+
Protein in Weight of flour taken
-
Grams
1
2
Water extract
First alcohol extract
Percent
Percent
4'
3 (1)
+ (2)
Second alcohol extract
Percent
Percent
0.19 0.18 0.17 0.17
5:lB 9.09 4.33 8.99 9 , 3.22 9.10 2.83 8.87 9 -Average = 9.01 Alcohol soluble protein A.O.A.C. method = 9.04 18
3.93 4.66 5.88 6.04
1s
TABLE I (Cont.) Weight of flour taken Grams
18 18 9 9
I
I
Protein in
5
6
,7
8
Third alcohol extract
95y0 alcohol ether extract
Total, protein extracted
Viscosity reading
Percent
Percent
Percent
M0
0.13 0.14 0.13 0.11
0.02 0.02
9.43 9.33 9.42 9.16 9.33
191 195
0.02 0.01 Average
=
...
...
Physico-Chemical Proflerties of Flours.
V.
677
agreement and the average agrees very well with the alcohol soluble protein determined directly according to the A . 0 . A . C meth0d.l The viscosity value is not as high as was found in previous determinations, i. e., 258” M for the 18 grams, where treatment with alcohol was omitted, yet the imbibition here produced is of a high order of magnitude. The results of this experiment indicated that most of the gliadin could be removed from the flour by simple extraction with distilled water. I n order to ascertain how far this was true, 18 gram portions of the flour were shaken with 500 cc of distilled water, the flour particles allowed to settle, and the supernatant liquid decanted into a 2 liter volumetric flask. The extraction was repeated three times more and the protein removed by the two liters of water was determined in an aliquot of the extract. Three samples of flour were thus extracted, the first 8 times, the second 12 times, and the third 16 times, with 500 cc portions of distilled water and the protein removed by the water determined. The remaining residues were each treated with 100 cc of 95 percent alcohol which with the water present in the residue made a final concentration of 70 percent alcohol and the material shaken in a mechanical shaker for 2 hours. The extract was removed by centrifuging and the residue was twice more extracted with 100 cc of 70 percent alcohol. The protein removed in these alcoholic extracts was then determined. The residues were air dried and made up to a total volume of 100 cc with distilled water and the maximum viscosity produced in the presence of lactic acid was determined. The results are given in Table 11. The viscosity values obtained are not as high as those in Table I yet about 1 percent more protein was removed in Table I1 which would roughly account for the difference. The results indicate that most of the alcohol soluble prote n can be removed by treatment with water, but even after extraction with 16 successive portions of 500 cc each of distilled “Methods of Analysis.” Association of Official Agricultural Chemists, Washington, (1920), p. 167-168.
Paul F . Sharp avzd Ross
678
A. Gortner
water approximately 1 percent of alcohol soluble protein remains. Some of the glutenin was undoubtedly removed with the water extract, either in solution or in the form of a suspension which had not settled out.
TABLE I1 18 gram portions of flour 1009 were repeatedly extracted with 500 cubic centimeter portions of water and the protein (N X 5.70) in the combined extract determined. The number of extractions is indicated in the table. The remaining residue was finally extracted three times with 100 cc portions of 70 percent alcohol. The protein content of these extracts was determined. The residue was made up to 100 cubic centimeters with water and the maximum viscosity in the presence of lactic acid was determined. The torsion wire used read 29.2' M with a 60 percent sucrose solution whose viscosity is 43.86 centipoise. Percentage protein in ~
First 4 Second 4 water extracts water extracts
Fourth 4 water extracts
....
1.61 1.56 1.43
6.67 6.77 6.77
I I1 I11
Third 4 water extracts
0.75 0.63
0.52
TABLE I1 (Cont.)
I
Percentage protein in Alcohol extracts after water extractior
I I1 I11
'
1.85 1.25 1.12
Total protein extracted
10.13 10.33 10.33
I -
Viscosity M"
. . . .* 157 149
* Residue lost by accident. On the theory that glutenin is solely responsible for the viscosity changes, the lower viscosity readings obtained in Tables I and I1 could not be accounted for entirely on the basis of the removal of some of the glutenin due to a slight solubility or mechanical dispersion in the water. It is well known that naturally occurring proteins are very sensitive t o changes in their environment and it was suspected that
Physico-Chemical Properties of Flours.
V.
679
this lower viscosity might be due to either to a denaturizing effect of the 70 percent alcohol or to the subsequent drying of the residue. Sharp and Gortnerl have shown that gluten cannot be dried in a vacuum oven at a temperature of 50" C without altering its colloidal properties. The above experiment was therefore repeated, except that, instead of drying after the alcoholic extraction, the alcohol was removed by 4 subsequent extractions with 500 cc of distilled water. The maximum viscosity values obtained were, for the 8 extractions 202" M, for the 12 extractions 160" M, and for the 16 extractions 105" M. These experiments indicated that it is the denaturization of the glutenin by the 70 percent alcohol which causes the lowered viscosity readings. The colloidal properties of the glutenin are thus markedly altered by treatment with 70 percent alcohol. Additional proof that this is the case is found in the next experiment. Three samples of flour of 18 grams each were extracted, the first 8 times with 500 cc of water, the second 12 times and the third 16 times, and the increase in viscosity as produced, by various additions of lactic acid then determined on the residue. It will be noted that this is a repetition of the preceding experiments with the exception that the treatments with alcohol are omitted. The results obtained are expressed graphically in Figure 1. T h e m a x i m u m viscosity reading, 456" ill, i s much higher than a n y of the previous values, yet practically all of the alcohol soluble protein has been removed. The effect of even 0.10 cc of normal lactic acid is very pronounced, causing an increase in viscosity of over 90 times the value obtained with distilled water (4" M to 365" M). This viscosity is reached almost instantaneously on the addition of the lactic acid. Loeb2 suggests that the physical behavior of proteins, such as swelling, viscosity changes, etc., which have been 1 P. F. Sharp and R . A. Gortner: "Physico-chemical Studies of Strong and Weak Flours, 11." Jour. Phys. Chem., 26, 101-136 (1922). 2 J. Loeb: "Proteins and the Theory of Colloidal Behavior," p. 119
(1922).
680
Paul F . Sharp and Ross A. Gortner
ascribed by earlier workers to the colloidal state of the proteins, is in reality due to the formation of definite chemical compounds with the acids, alkalies and salts; and that the hydration and viscosity phenomena are due to the osmotic behavior of the protein ions in accordance with the Donnanl equilibrium and the colloidal condition, per se, does not enter into the swell__
The effect of repeated extraction of the flour with water on the viscosity as influenced by lactic acid and the effect of adding salts to the system
ing or viscosity changes. Loeb has presented a mass of data in support of his view. It is not our intention to seriously discuss this question in the present paper. We wish merely t o point out one or two pertinent facts which may have a bearing upon the question. I n the first place the argument that proteins, such as egg albumen, are known to occur in monomolecular solution (cf. the studies of Sorensen et a1.)’ does not in our opinion 1
2
F. G. Donnan: Zeit. Elektrochem., 17, 572-581 (1911). S. P. L. Sorensen, et al.: “Studies on Proteins,” Comptes rendus. Lab.
Carlsberg, 12, 1-372 (1915-1917).
Physico-Chemical Properties of Flours.
V.
68 1
preclude the possibility that such solutions may possess purely colloidal reactions. The colloidal state has been defined as depending upon the size of the particle, those particles between ~ 1pp being within the colloidal realm. the diameter of 0 . 1 and Apparently many workers have read into this definition the supposition that such particles must be composed of aggregates of molecules. Such a supposition is apparently not true. Robertson’ has calculated the diameter of the casein molecules on the assumption that the molecular volume is an additive function of the volume of the constituent atoms. He concludes that the diameter of the casein molecule must ~ ~ that the formula is C777H1241 N1970250be about 2 . 4 providing S4P4with a molecular weight of approximately 17,000. Even in monomolecular solutioii, a particle of such size should show surface phenomena characteristic of the colloidal state. T h e casein molecule, however, i s undoubtedly hydrated in solution so that the act.tlal diameter of the particle i n solution must considerably exceed the minimum diameter of 2. ~ p p . Ultrafiltration data also indicate that the casein particle as it exists in solution has a relatively large diameter considerably exceeding 2 . 4 ~ . Whether or not casein exists in solution in monomolecular aggregates is still unknown. Sorensen, however, has shown that egg albumen solutions are definitely monomolecular solutions which rigidly obey the equilibria demanded by the phase rule. He concludes from direct osmotic pressure measurements that the molecular weight of egg albumen is of the order of 34,000. Ultrafiltration experiments again indicate that these molecules are highly hydrated and are of colloidal dimensions. Such solutions undoubtedly show chemical reactions, but we belieue that they must also show surface reactions characteristic sf similarly sized particles in the colloidal state. Turning again to Figure 1, we note that 0.1 cc of normal lactic acid increased the viscosity of the flour suspension from T. B. Robertson: “The Physical Chemistry of the Proteins,” pp. 343-4 (1918). 2
S. P. I,. Sorensen: loc. cit.
682
Paul F . Sharp and Ross A . Gortner
4" MacMichael to 365" M. This increase in viscosity was practically instantaneous (within 2 or 3 seconds!) and on the addition of 0.5 cc of normal MgS04 solution the viscosity fell practically instantaneously from 439" M to 11 M. The rate of viscosity change and the extreme lability of the systems suggest very strongly that we are dealing with surface reactions characteristic of the colloidal realm, rather than chemical combinations and the subsequent osmosis of the Donnan equilibrium. The real test of Loeb's view that all osmotic and viscosity changes are due to the Donnan equilibrium will come when some one applies it to carbohydrate solutions, With pentosan solutions we have as great viscosity values as are found in protein solutions, nevertheless no one has shown that such gums and mucilages combine with acids and basis in stoichiometrical relationships. The depressing effect of salts on the viscosity of a flour suspension hydrated by lactic acid is very pronounced as can be seen from Figure 1. The depressing effect of magnesium sulfate is greater than that of magnesium chloride. This is in agreement with the effect of these two salts on the flours which have not been extracted with water.l These experiments indicate that the glutenin is the protein mainly responsible for the increase in imbibition produced by acids acting on flour-in-water suspensions. This conclusion that glutenin is the protein responsible for the imbibition changes between strong and weak flours was reached late in the fall of 1921 and all of the experimental work was completed prior to May 1922. Woodman2 in a recent paper, and using entirely different methods, reaches a similar conclusion that the difference between a strong and weak flour is due to a difference in the glutenin fraction. Woodman bases his evidence on the optical behavior of the pure flour proteins when racemized at 37" by dilute alkali. He finds the gliadin from both types of flour to be identical, but the natural glutenins R. A. Gortner and P. F. Sharp: "The Physico-Chemical Properties of Strong and Weak Flours, IV." Jour. Phys. Chem., 27, 567 (1923). 2 H. E. Woodman: Jour. Agr. Sci., 12, 231-243 (1922).
Physico-Chemical Properties of Flours.
V,
683
possess different specific optical rotations and, when treated with alkali, show different racemization curves. Woodman states “The factor which determines the shape of the loaf and which appears to be directly related to the physical properties of the gluten of the flours, is possibly dependent on the particular glutenine mechanism possessed by the wheat. The results of this investigation suggest that the strong wheat synthesizes one type of glutenine and the weak wheat a different type.” Such a conclusion we regard as strongly confirming our experiments as recorded in the present series of papers. Woodman suggests that if the physical properties of strong and weak flours are determined by the glutenin fraction it ought, theoretically, to be possible to prepare a “strong” gluten by mixing the gliadin from a weak flour with the glutenin from a strong flour. He was, however, unsuccessful in such an experiment and believes that his failure was due to two factors (1) “It is not possible by grinding the proteins together to effect the same intimacy of mixture as occurs naturally in the flour” and (2) “The physical properties of the proteins themselves have probably been considerably modified during the process of their isolation as a result of prolonged contact with different reagents (alcohol, ether, alkali etc.).” Here again his second conclusion admirably sustains our findings that treatment of the glutenin with alcohol markedly changes its physical properties. This conclusion we expect to enlarge upon in the seventh paper of this series.
Summary 1. Glutenin is the protein mainly responsible for the increase in imbibition when flour-in-water suspensions are acted on by acids. 2. A large proportion of the gliadin can be removed from flour by extraction with distilled water while very little of the glutenin is removed by such treatment. 3. The colloidal properties of the glutenin as measured
684
Paul F . Shayfl alzd Ross A. Gortlzer
by changes in rate of imbibition and maximum imbibition capacity are markedly altered by treatment with alcohol of a relatively high concentration. 4. A “strong” flour is apparently characterized by the presence of a glutenin possessing marked colloidal properties whereas the corresponding colloidal properties of the glutenin in a weak flour are much less pronounced.
