Electrophoretic Separation and Determination of the Amino Acid Composition of Cereal Crop Proteins

V. M. Kolesov

(Presented by Prof. L. P. Kulev, Doctor of Chemical Sciences)

One of the most pressing tasks of modern organic chemistry is to elucidate the internal structure of protein substances. In view of the enormous size of protein molecules, the use of purely chemical methods of investigation to determine the structure of the latter is becoming clearly insufficient. It is absolutely necessary to employ, for this purpose, not only biochemical and chemical methods but also physical and physicochemical methods of investigation, which are beginning to acquire exceptionally great importance in modern chemistry.

Proteins of plant origin, in particular the proteins of cereal crops, still remain insufficiently studied with respect to their physicochemical properties, and there is no general point of view whatsoever in this area. In our investigations [1] it was shown that, according to indices of physicochemical characterization—relative viscosity, surface tension, isoelectric point, specific rotation, binding of hydrogen ions, and the amount of total nitrogen and amine nitrogen—the protein preparations studied show a tendency to converge with one another. This circumstance gives grounds for suggesting the presence, in all probability, of a single spatial configuration underlying the structure of the above-mentioned proteins.

A further development of these investigations was the need to subject our protein preparations to electrophoretic separation and to study them from the standpoint of their amino acid composition.

The study of the amino acid composition in prolamins and glutenins may serve as a basis for further investigation in the field of the structure of these proteins, chiefly in determining the number of peptide chains, with subsequent establishment of the nature of the N- and C-amino-acid residues in the molecules of these proteins.

Material and Methods

The objects of investigation were the following preparations of protein substances: rye gliadin, wheat gliadin, barley hordein, oat avenin, and couch-grass pyrein. Protein preparations of the prolamin group were ...

were obtained by the method of T. Osborne [2], as modified by A. Kiesel [3]. For the extraction of wheatgrass, the procedure described by V. L. Kretovich [4] was used. The gluten was washed by the method of P. N. Shibaev [5]. Barley hordein was extracted from the gluten of the latter.

The amino-acid composition of the prolamins of cereal crops, despite the long period during which their chemical composition has been studied, has still not been investigated sufficiently fully. In the lists of amino acids characterizing the composition of wheat gliadin in the monograph by R. Block and D. Bolling [6], phenylalanine, serine, and valine are absent. In its chemical composition, the grain of barley and oats approaches the grain of wheat and rye. However, the data characterizing the amino-acid composition of these cereals are very limited. In the tables of the amino-acid composition of proteins given in the textbook by N. P. Kozmina and V. L. Kretovich [7], there are no data on the presence in wheat gliadin, hordein, and avenin of such important amino acids as glycocol, threonine, serine, and methionine. According to the studies of T. Osborne and C. Clapp [8, 9], the presence of valine, threonine, methionine in rye gliadin and of threonine and methionine in wheat glutenin was not established. However, the data relating to this period of protein research have become considerably obsolete. In connection with the work of N. V. Tsitsin [10] on the problems of wheat–wheatgrass hybrids and the discovery by some investigators [16, 17] of gluten in wheatgrass seeds that is not inferior in its baking qualities to wheat gluten, interest arose in studying the amino-acid composition of wheatgrass prolamin—the alcohol-soluble fraction of wheatgrass-seed proteins. V. L. Kretovich [4] reports only on the content of two amino acids—histidine and arginine—present in wheatgrass. In view of the foregoing, a study was undertaken of the amino-acid composition of the above-mentioned proteins by means of two-dimensional paper partition chromatography [11, 12]. The presence of tryptophan in the proteins under study was confirmed in a separate test according to Forth [13].

Results

The results of the determination of the amino-acid composition are given in Table 1. On the basis of repeatedly obtained chromatograms it was possible to establish the presence of 15 spots and to identify, by the \(Rf\) values of standards, 15 different amino acids (Fig. 1).

Analyzing the results of the determination of the amino-acid composition of the studied proteins, it should be noted that, in terms of the quantitative content of many amino acids, the protein preparations can apparently be divided into approximately three subgroups. The first subgroup may include the gliadins of rye and wheat and wheatgrass pyrein. Hydrolysates of rye and wheat gliadin contain the same amino acids, but in a different quantitative ratio. The difference between them is observed in the amounts of glycocol, alanine, serine, proline, and tryptophan. In terms of the quantitative content of most amino acids, wheatgrass pyrein approaches rye gliadin.

The second subgroup consists of avenin and hordein. When comparing the quantitative content of certain amino acids present in samples of avenin and hordein with the content of these amino acids in the related prolamins of wheat, rye, and wheatgrass, attention is drawn to the elevated content, relative to gliadins and pyrein, of glutamic acid—twice exceeding the content of phenylalanine—and the somewhat reduced content of threonine, alanine, and valine. Cha

A characteristic feature of this subgroup is the almost equal amount, in both proteins, of leucine, glutamic acid, phenylalanine, glycocoll, and threonine, and the fairly similar content of the other amino acids, with the exception of lysine, serine, proline, and the absence of tryptophan in oats.

Fig. 1. Chromatogram of a hydrolysate of rye glutenin.
From right to left: pyridine—acetic acid; from top to bottom: butanol—acetic acid:
1—cystine, 2—lysine, 3—histidine, 4—arginine, 5—aspartic acid, 6—glycocoll, 7—serine, 8—glutamic acid, 9—threonine, 10—alanine, 11—proline, 12—tyrosine, 13—valine (methionine), 14—phenylalanine, 15—leucine.

Rye glutenin and wheat glutenin may be assigned to the third subgroup. From the data on determination of the amino-acid composition of rye and wheat glutenins it follows that, like the prolamins considered above, they contain the same amino acids, but differ somewhat from them in the quantitative ratio of the latter. Attention is drawn to the great similarity in the level of content, in both glutenins, of valine, leucine, glutamic acid, and tryptophan. In the content of the other amino acids there is a considerable difference.

Because of the identical \(Rf\) value of valine and methionine and the easy oxidation of the latter during hydrolysis in 20% HCl, the presence of methionine could not be established with certainty.

Electrophoretic separation of the protein preparations was carried out on a Tiselius microapparatus. The proteins were dissolved in 0.1 N acetic acid with pH 2.85 and ionic strength 0.1. One-percent solutions of the protein preparations were dialyzed against a solution of 0.1 N acetic acid and then subjected to electrophoretic analysis.

Electrophoresis was carried out for 1–3.5 hours at a resistance of 45,000–125,000 ohms, a voltage of 250–350 V, and a current of 0.4–2.4 mA.

Table 1

Amino-acid composition of protein preparations

It was shown that wheat gliadin No. 1, wheat gliadin No. 2, rye gliadin No. 2, pyrein, and hordein consist of four fractions; rye gliadin No. 1, rye gliadin No. 3, hordein No. 2, and avenin consist of three fractions (Fig. 2). Similar results for wheat gliadin were obtained by E. I. Medvedeva [14] and by Miles [15] during electrophoresis on paper and on the Tiselius–Svensson apparatus.

Conclusions

By the method of partition chromatography on paper, amino-acid residues of glycocol, alanine, leucine, lysine, histidine, arginine, glutamic acid, cystine, proline, tyrosine, threonine, serine, phenylalanine, valine, and aspartic acid were found in the proteins studied.

In addition to the structural elements of gliadin known from the literature, valine was detected for the first time in the hydrolysate of rye gliadin.

Fig. 2. Electropherograms of protein preparations.
1 — rye gliadin No. 2; 2 — wheat gliadin No. 1; 3 — rye gliadin No. 3; 4 — hordein No. 2; 5 — wheat gliadin No. 2; 6 — rye gliadin No. 2; 7 — pyrein; 8 — hordein No. 1; 9 — avenin; 10 — rye gliadin No. 1.

(+ methionine), threonine, cystine, and lysine. For the first time, the amino-acid composition of wheatgrass was also established by the method of partition chromatography on paper; the presence was ascertained and the quantitative content determined of threonine and serine in avenin, and of tryptophan in wheat glutenin and rye glutenin.

On the basis of the quantitative determination of many amino acids, it may be concluded that the chemical nature of wheat, rye, wheatgrass, hordein, and avenin gliadin is similar.

By the classical method of electrophoresis in the Tiselius apparatus it was shown that preparations of prolamins, being heterogeneous, consist of four fractions (wheat gliadins No. 1 and No. 2, rye gliadin No. 2, hordein No. 1, wheatgrass), while some of them (rye gliadins No. 1 and No. 3, avenin, hordein No. 2) consist of three fractions, which also makes it possible to speak of the close chemical nature of the proteins investigated.

REFERENCES

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