STABILITY OF TERTIARY STRUCTURE OF PHASEOLIN OF RED KIDNEY BEAN (PHASEOLUS-VULGARIS) AS LIMITING FACTOR IN PROTEOLYSIS

The major storage protein, phaseolin, of red kidney bean (Linden variety) was purified by ammonium sulfate fractionation between 3 and 4.1 M. It was composed of three subunits with MW 49,000, 45,000, and 42,000 and there were no disulfide bonds. Phaseolin was treated with heat (121-degrees-C, 15 min), pH (1.0, 7.5, 9.0), urea (2 to 10 M), guanidine (3 to 8.5 M) and alkali (0.02 N NaOH) at 35-degrees-C to destabilize the tertiary structure. Increase of in vitro proteolysis by the prior treatments was estimated with chymotrypsin, trypsin, pepsin and pronase. Native phaseolin was more resistant than Hammarsten casein to hydrolysis by chymotrypsin, trypsin, pepsin and pronase. An equimolar mixture of chymotrypsin and trypsin gave lower proteolysis than the sum of hydrolysis by each proteinase separately. Chymotrypsin hydrolysis products of phaseolin inhibited trypsin, and trypsin hydrolysis products of phaseolin inhibited chymotrypsin. All the treatments listed above enhanced in vitro proteolysis. Phaseolin treated with 0.02 M NaOH had higher hydrophobicity and higher absorbance between 240 and 350 nm than native phaseolin, indicating that its tertiary structure was destroyed. Autoclaving at pH 7.5, treatment with 8.5 M guanidine or with 0.02 M NaOH increased the extent of proteolysis of phaseolin to a similar or higher level than Hammarsten casein. Guanidine (8.5 M) destroyed the tertiary structure of phaseolin sufficiently that almost all susceptible bonds were hydrolyzed by pepsin, chymotrypsin and trypsin (and probably pronase).