Natural plant enzyme inhibitors. Isolation of a trypsin/α‐amylase inhibitor and a chymotrypsin/trypsin inhibitor from ragi (Eleusine coracana) grains by affinity chromatography and study of their properties

Abstract

AbstractA trypsin/α‐amylase inhibitor (TAI) and a chymotrypsin/trypsin inhibitor (CTI) were isolated in homogeneous forms from ragi grains by affinity chromatography using immobilised chymotrypsin and immobilised trypsin. Both the inhibitors were capable of inhibiting the caseinolytic and amidolytic activities of bovine trypsin whereas the esterolytic activity of the enzyme was only weakly affected. While TAI had no action on chymotrypsin, the CTI exerted an inhibitory effect on the caseinolytic activities of bovine α‐, β‐, γ‐ and ş‐chymotrypsins. Both the inhibitors could inactivate the proteolytic actions of bovine as well as human crude pancreatic preparations, TAI showed inhibitory activity against human pancreatic, porcine pancreatic and human salivary amylase in the ratio of 6.5:5:1. The possible practical application of TAI for the purification of α‐amylases by affinity chromatography is indicated based on the demonstration of the dissociation of porcine pancreatic amylase from a ‘trypsin‐TAI‐amylase’ trimer complex in the presence of maltose. The antichymotryptic activity of CTI was less stable than its antitryptic activity at high temperature, acidic pH and on treatment with pepsin. Modification of arginine residues by 1,2‐cyclohexane dione led to a preferential inactivation of its antitryptic activity. Treatment of CTI with trinitrobenzene sulphonic acid or pronase, however, caused almost identical loss of both antitryptic and antichymotryptic activities. The mode of inhibition of CTI was non‐competitive for trypsin but was uncompetitive for chymotrypsin. The CTI could bind to immobilised trypsin or chymotrypsin but not to immobilised TPCK‐chymotrypsin or chymotrypsinogen.