| Determination of wheat flour proteins by gel electrophoresis John M. LAWRENCE and D. R. GRANT Department of Agricultural Chemistry, Washington Agricultural Experiment Stations, Washington State University, Pullman, Wash., U. S. A. Many of the differences in the quality of wheat varieties for various food purposes have been suggested to be due to more or less subtle differences in the proportions of individual proteins in the flour (1). Clearly the effort to breed wheat for quality would be much advanced by expeditious methods for quantitative determination of the proteins. Among the newer methods for protein separations, zone electrophoresis in starch gels (2,3) and polyacrylamide gels (4,5) have shown the most resolving power, but their application to quantitative analytical procedures has not been extensively studied. The present method makes use of an aluminium lactate buffer extract of flour (pH 3.2, ionic strength 0.1). A single thorough extraction followed by ultracentrifugation yields a clear extract which contains 50 to 80% of the flour proteins. Polyacrylamide gels were used in a specially designed apparatus such that electrophoresis occurs in a vertical orientation in individual water - cooled strips for each sample. The sample volumes introduced into slots in the gels are accurately measured. The gels are cast in water with or without urea. (Urea improves the resolution, but causes minor changes in the pattern). Aluminium lactate buffer is then introduced to the gel by overnight electrophoretic equilibration. A two - hour electrophoresis of the sample spreads the pattern well throughout the length of the gel strip, though a longer run provides better separation of the predominant slower -moving proteins. The proteins are stained with amido black dye, and the absorbance of the bands is determined by passing the strip through a Photovolt densitometer. The true protein content of individual bands (or groups of bands) is calculated by multiplying the peak areas by correction factors for the dyebinding capacities of the proteins. These were determined by cutting out the bands from the gels, hydrolyzing, and analyzing for the total amino nitrogen content as a measure of the true protein concentration. It was found that the slower moving proteins generally bind less dye than the faster ones. The two fastest bands in particular represent very little protein. As many as 30 different bands have been detected on such gel strips, though many of these are not sufficiently resolved for separate quantitative determination. Generally about eight different groups of bands have been determined Quantitative differences have been found in the relative protein make - up of different market classes of wheat. Figure 1 shows some striking differences among some varieties tested. Differences between air - classification fractions which had not been revealed by previous studies (6), were shown by this method. This work was largely supported by a contract with the U. S. Department of Agriculture, authorized by the Research and Marketing Act of 1946, and supervised by the Western Utilization Research and Development Division of the Agricultural Research Service. The authors wish to express their appreciation to Mr. N. K. Patni and Mrs. Hedwig Herrick for technical assistance, and to Dr. C. F. Konzak and Mr. R. K. Bequette for wheat flour samples to test the applicability of the method. List of References (1) Pamela M. BELL and D. H. SIMMONDS, Cereal Chem. 40, 121-8 (1963). (2) G. A. H. ELTON and J. A. D. EWART, J. Sci. Food Agr. 13, 62-72 (1962). (3) Janet S. D. GRAHAM, Austral. J. Biol. Sci. 16, 343-9 (1963). (4) J. W. LEE, Biochem. Biophys. Acta 69, 159-60 (1963). (5) C. C. NIMMO, Mary T. O'SULLIVAN, A. MOHAMMAD, and J. W. PENCE, Cereal Chem. 40, 390-8 (1963). (6) C. W. WRIGLEY, J. Sci. Food Agr. 14, 120-4 (1963). |
| * Present address: Department of Chemistry, University of Saskatchewan, Saskatoon Saskatchewan, Canada |