Isolation and Characterization of the Subunits of Phaseolus vulgaris α-Amylase Inhibitor

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Abstract
An α-amylase inhibitor (PHA-I) of the white kidney bean (Phaseolus vulgaris) was found to be composed of two kinds of subunits and they were isolated on a size-exclusion column by HPLC under denaturing conditions. The α-subunit was free from tryptophan and cysteine and the β-subunit contained no methionine or cysteine. There was no marked resemblance in tryptic peptide map between these subunit polypeptides. The a-subunit contained 28% by weight of carbohydrate, mainly made up of high mannose-type oligosac-charides, whereas the sugar moiety of the β-subunit amounted to 7% by weight and seemed to be predominantly composed of xylomannose-type oligosaccharides. By SDS-PAGE following deglycosylation, the molecular weights of the polypeptides of α- and β-subunits were shown to be 7,800 and 14,000, respectively. These values were consistent with molecular sizes obtained for α- and β-subunits by gel permeation HPLC in 6 M guanidine hydrochloride. The molecular weight of the native PHA-I, 28,800, obtained by gel permeation HPLC under non-denaturing conditions, suggested a heterodimeric structure for PHA-I.

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