KINETIC AND STRUCTURAL STUDIES ON THE INTERACTION OF PROTEINASE-INHIBITOR FROM DOLICHOS-BIFLORUS (HORSE GRAM)

One of the major components of multiple inhibitors from Dolichos biflo rus (horse gram) (HGPI) inhibits both bovine trypsin and chymotrypsin, resembling other Bowman-Birk-type protease inhibitors. Ultraviolet ab sorbance 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 (lambda(max) 336 nm) is due to the tyrosine residues in the hydrophilic environmen t. Disulfide linkages and tyrosine residues contribute to the near-ult raviolet circular dichroism spectra. Far-ultraviolet circular dichrois m measurements indicate the absence of any helical structure, 31% beta structure, and the rest aperiodic structure. Aromatic amino acids are involved in the interaction of the inhibitor with trypsin or chymotry psin. The equilibrium constants for the interaction of HGPI with chymo trypsin/trypsin were 2.9 x 10(5) and 5.2 x 10(5) M(-1), respectively, with an estimated stoichiometry of 1:1 with either of the enzymes.