DOUBLE-HEADED TRYPSIN/CHYMOTRYPSIN INHIBITORS FROM HORSE GRAM (DOLICHOS BIFLORUS): PURIFICATION, MOLECULAR AND KINETIC PROPERTIES

Abstract

Four proteinase inhibitors were purified to homogeneity from horse gram (Dolichos biflorus). These inhibitors are double-headed and inhibit trypsin and chymotrypsin simultaneously and independently. Dissociation constants range between 0.87 and 4.6 × 10−7 M. Each of the four isoinhibitors possesses a crucial lysine residue at the trypsin reactive site. These inhibitors have molecular masses of 8.5 kDa and isoelectric points of 4.6 to 5.6. They exist mainly as dimers under physiological conditions. Amino acid analysis revealed high levels of half-cystine, serine, aspartate and proline but low levels of methionine and aromatic amino acids. Amino-terminal sequence analysis revealed that each of the four isoinhibitors have a conserved core sequence but are divergent at the N-terminal end. These inhibitors belong to the Bowman-Birk (BBI) family of proteinase inhibitors as reflected by their inhibitory properties, amino acid composition and homology to other BBIs.