Kinetic and structural studies on the interaction of proteinase inhibitor from Dolichos biflorus (horse gram)

Abstract

One of the major components of multiple inhibitors from Dolichos biflorus (horse gram) (HGPI) inhibits both bovine trypsin and chymotrypsin, resembling other Bowman-Birk-type protease inhibitors. Ultraviolet absorbance measurements indicate the presence of two tyrosine residues, the absence of tryptophan, and the dominance of disulfide linkages in the molecule. The intrinsic fluorescence emission maximum (Amax 336 nm) is due to the tyrosine residues in the hydrophilic environment. Disulfide linkages and tyrosine residues contribute to the near-ultraviolet circular dichroism spectra. Far-ultraviolet circular dichroism measurements indicate the absence of any helical structure, 31% 13 structure, and the rest aperiodic structure. Aromatic amino acids are involved in the interaction of the inhibitor with trypsin or chymotrypsin. The equilibrium constants for the interaction of HGPI with chymotrypsin/trypsin were 2.9 x 105 and 5.2 x 105 M-1, respectively, with an estimated stoichiometry of 1:1 with either of the enzymes.