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Phaseolin differs from other native legume proteins in that its hydrolysis by trypsin and pepsin stops after a limited number of peptides have been cleaved off. Concomitantly, trypsin splits each phaseolin subunit approximately into halves. The N-terminal sequencing of these final hydrolysis products of high molecular mass showed loss of a tetrapeptide from the N terminus of each subunit, with a second cleavage in the interdomain linker. Other probable sites cleaved by trypsin, which account for the quantity of degraded protein, may be deduced from the tertiary structure of phaseolin and from the specificity of trypsin. Proteolysis by pepsin is limited to cleavage of seven amino acids from the N terminus, and of two to three peptides probably from the disordered C-terminal segment of phaseolin subunits. The cleavage site in the N-terminal sequence has been identified. The peculiarities of the phaseolin structure that may cause its resistance to the proteolytic attack are discussed. Keywords: Phaseolin; proteolysis; pepsin; trypsin

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Two types of cysteine proteases, low-specificity enzymes from the papain family and Asn-specific from the legumain family are generally considered to be the major endopeptidases responsible for the degradation of seed storage proteins during early seedling growth. The action of the corresponding enzymes (CPPh1 and LLP, respectively) from common bean (Phaseolus vulgaris L.) on phaseolin (the common bean storage protein), and on the homologous soybean (Glycine max (L.) Merrill) storage protein, beta-conglycinin, was studied. Under the action of LLP, proteolysis of phaseolin was limited to cleavage of its interdomain linker. No cleavage of the interdomain linker occurred in beta-conglycinin with LLP. LLP action was restricted to splitting off the disordered N-terminal extensions of alpha and alpha' subunits. No extensive hydrolysis (degradation to short TCA-soluble peptides) of either protein occurred under the action of LLP. CPPh1 cleaved the phaseolin subunits into roughly half-sized fragments at the onset of proteolysis. The cleavage was accompanied by a small (8-10%) decrease of protein. No decrease of protein occurred with further incubation. Thus the two most active proteinases detected in common bean seedlings individually were incapable of the extensive degradation of phaseolin. Extensive hydrolysis of phaseolin was only achieved by the consecutive action of LLP and CPPh1. Similar cleavages occurred during the action of CPPh1 on beta-conglycinin. However, by contrast with phaseolin, CPPh1 by itself accomplished the extensive hydrolysis of beta-conglycinin. The differences in the course of proteolysis of the proteins studied were determined by their structural peculiarities.