CHARACTERIZATION OF THE SUBUNITS OF BETA-CONGLYCININ, AND APPLICATION OF THE ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) TO THE DETERMINATION OF THE CONTENTS OF BETA-CONGLYCININ AND GLYCININ IN SOYBEAN (GLYCINE MAX (L.) MERRILL) SEEDS
Abstract
Four polypeptides were isolated from (beta)-conglycinin prepared from soybean cultivar CX 635-1-1-1. They were the (alpha), (alpha)', (beta) and (gamma) subunits. Electrophoretic analysis of the cyanogen bromide cleavage fragments from subunits (alpha), (alpha)' and (gamma) showed that only a few fragments were produced. This was consistent with their low methionine content. The (beta)-subunit had no methionine and was not cleaved by cyanogen bromide. The amino acid composition of the subunits revealed that the most abundant amino acids in all subunits were glutamate plus glutamine, and aspartate plus asparagine, followed by serine, proline, leucine and arginine. The NH(,2)-terminal amino acid sequences were different for each subunit, although (alpha) and (alpha)' were homologous. The NH(,2)-terminal amino acid sequence analysis of the two cyanogen bromide fragments found showed that the large fragment (CB-1) had the same sequence as the uncleaved (alpha) subunit, while the small fragment (CB-2) had a different one, and therefore originated from the interior of the molecule. Antibodies were raised against purified (beta)-conglycinin and glycinin from soybean seeds. The purified antibodies were shown to be monospecific and were used to test the feasibility of employing ELISA to quantitation of these two storage proteins in the soybean seed. The useful concentrations of antibody, and antibody-alkaline phosphatase conjugate used in ELISA were determined in two solid support systems, and the microELISA('R) plates and the Gilford('R) EIA cuvettes. Also the range of dilutions from crude soybean seed extracts appropriate for the quantitation of (beta)-conglycinin and glycinin were determined. Seeds of 32 soybean cultivars were analysed for total protein, as well as their (beta)-conglycinin, and glycinin contents. Total seed protein content varied from 39.5 to 55.5% (mean (+OR-) S.D. = 45.3 (+OR-) 3.0). The (beta)-conglycinin content ranged between 7.3 and 16.1% (mean (+OR-) S.D. = 11.0 (+OR-) 2.6) of the seed dry weight, while glycinin content was between 9.8 and 26.7% (mean (+OR-) S.D. = 17.5 (+OR-) 4.2). The two proteins together accounted for 37.7 to 88.2% (mean (+OR-) S.D. = 63.1 (+OR-) 13.2) of the total seed protein. The glycinin to (beta)-conglycinin ratio varied from 1.10 to 2.91 (mean (+OR-) S.D. = 1.63 (+OR-) 0.37).
Degree
Ph.D.
Subject Area
Botany
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