Quantitative Analysis of Soybean Proteins

Cereal Chem. 63(6):493-496 Quantitative analyses of soybean globulins were made by densitometry Proteins extracted with both water and 0.5M sodium chloride contained in a sodium dodecyl sulfate (SDS)-polyacrylamide slab gel using lysozyme glycinin (36.5%), /3-conglycinin (27.8%), y-conglycinin (6.2%), Kunitz as an internal standard protein and Coomassie Brilliant Blue as a staining trypsin inhibitor (2.9%), basic-7S globulin (3.0%), and other proteins reagent. The slopes of standard curves for soybean globulins showed a (23.6%) that included whey protein. The relative concentrations of the considerable difference, depending on their proteins. The densitometric major components (glycinin and /3-conglycinin) were evaluated to intensity for the 7S globulins of y-conglycinin, basic-7S globulin, and accuracies better than ± 15%, and the other minor components were f3-conglycinin was higher than for glycinin and Kunitz trypsin inhibitor, evaluated to ±20%. Polyacrylamide gel electrophoresis (PAGE) is a superior MATERIALS AND METHODS technique presently available for separating proteins, and Coomassie Blue is a sensitive protein stain. Much recent work in Materials quantitative PAGE is concerned with stains and staining Defatted soybean meal was prepared (Glycine maxvar. Raiden, procedures. Coomassie Blue has been reported to follow Beer's law from 1982 and 1983 crops in Iwanuma, Miyagi, Japan). The over only a low concentration range (Chrambach et al 1967, soybean seeds were dehulled, flaked, and defatted with hexane. Fazekas de St. Groth et al 1963), and there are difficulties in The/3-conglycinin (7S globulin), glycinin (llS globulin) (Thanh accurately determining the amount of protein within a gel. and Shibasaki 1976), y-conglycinin (7S globulin) (Sato et al 1984), Fishbein (1972) has shown the major sources of error in and basic-7S globulin (Yamauchi et al 1984) were isolated by the quantitative densitometry. cited methods. All the proteins were pure, as checked by column In this study, we adopted an internal standard method for chromatography and PAGE. Kunitz trypsin inhibitor was quantitative densitometry on PAGE. This method is not necessary purchased from Miles Laboratories (Pty) Ltd. Lysozyme and to accurately determine the amounts of protein loaded on a gel, but bovine serum albumin were obtained from Sigma Chemical Co. to determine the proportion of different protein components in a All reagents were of the highest grade. mixture. The quantities of some soybean globulins were reported from PrtiDermnio Brigs (956)and The yields of waterand NaCl-soluble proteins were determined ultracentrifugal analysis by W olf and by Lo rysie hog( ow y tal 19 1)a5r fe re)t ov ne se u immunological analysis by Koshiyama and Fukushima (1976). b or' ehd(or ta 91 srfre obvn eu Recetly HuhesandMurhy (983 an Muphyand albumin. For the standard curves of the peak area, the Resurreccion (1984) investigated the major soybean globulins by cnetain flszm n oba rtiswr ae analysis of eluted Coomassie Blue dye and an immunological gravimetrically. method, respectively. Ultracentrifugal analysis cannot distinguish Sml r rto /3-conglycinin from y-conglycinin. The immunological method Sapl Prparto rqieanindividual antibody, and one sample can measure only The waterand NaCl-soluble fractions were prepared by the one protein species. Densitometric analysis on PAGE can analyze moiedpcduefHuadEn(98)Fi.).Date all the proteins in one measurement. This paper describes the soybean meal was agitated with 20 volumes (v/w) of distilled water quanitaive ana ysis for not onl the maj r p ote ns g yci in liS for 1 hr at room tem perature and centrifuged ( 15,000 X g, 5° C, 25 globulin) and f3-conglycinin (7S globulin) but also the minor m).Tepcitaewsxrcedihdsildwtrinheae proteins -y-conglycinin (7S globulin), basic-7S globulin, and way three times. The waterand NaCl-soluble fractions were Kunitz trypsin inhibitor, dialyzed against water and lyophilized after measurements of each volume and the protein content. Acid-precipitated protein was obtained from the first water-soluble fraction adjusted to pH 4.8 ' Department of Food Chemistry, Faculty of Agriculture, Tohoku University, Sendai and dialyzed against water and lyophilized.