Chemical modification and complex formation studies with jack bean proteinase inhibitor.

Pretreatment of the purified jack bean inhibitor with enterokinase activated human pancreatic preparation for 1 hr decreased its inhibitory capacity against crystalline bovine alpha-chymotrypsin by 30% but did not affect its trypsin inhibitory activity. Preincubation of the inhibitor with bovine chymotrypsin for 60 min resulted in partial loss of the inhibitory potency. Complex formation studies by gel chromatography on Sephadex G-100 indicated that the trypsin-inhibitor and chymotrypsin-inhibitor complexes dissociated to release inactivated inhibitor and active proteinases. Gel chromatography of the inhibitor in presence of 1.5 M ammonium sulphate indicated that the inhibitor showed a tendency to aggregate without loss of biological activity. However, in 4.2 M salt medium after 3 hr, antichymotryptic activity was lost completely without any effect on antitryptic activity. Treatment with methylamine, a nucleophile, caused a greater loss of antichymotryptic activity. Trinitrobenzene sulphonate and ethylacetamidate, the amino group modifiers, affected only the antichymotryptic activity. Treatment with ninhydrin, a specific arginine modifier, at pH 9.0 abolished the antitryptic activity whereas only 50% of the antichymotryptic activity was lost. Diethylpyrocarbonate, a histidine reagent, also decreased only the antitryptic activity. Modification of tryptophan and cysteine residues of the inhibitor had no effect on its inhibitory potency. Treatment with mercaptoethanol and sodium borohydride caused nearly 50% loss of antitryptic and antichymotryptic activities. Chloramine-T, a reagent that modifies methionine residues, inactivated the inhibitor.

Natural plant enzyme inhibitors: isolation and properties of a trypsin inhibitor from jack bean (Canavalia ensiformis).

A protease inhibitor which is equally active on bovine and porcine trypsins was isolated in a homogenous form from jack bean (Canavalia ensiformis). The preparation with a molecular weight of 18 kDa was found to be a glycoprotein with a high half cysteine content. Isoleucine and tyrosine were found to be absent. The inhibitor was heat-stable and stable at pH 2.0 and 11.0. It was ten times less active on bovine alpha-chymotrypsin and pronase than on trypsin. It displayed weak action on subtilisin BPN, porcine elastase and pepsin. The inhibitor was most effective in blocking the total proteolytic, tryptic and chymotryptic activities of rabbit pancreatic preparation. The relative ratios of inhibitions of the three activities on rabbit, bovine and human systems were respectively 1250:100:1, 600:100:1 and 46:18:1. While different substrates (except denatured serum albumin) did not significantly alter the magnitude of inhibition of bovine trypsin, the extent of inhibition of bovine alpha-chymotrypsin by the jack bean inhibitor was highly dependent on the substrate used in the assay.