Purification and characterization of a proteinase inhibitor from field bean, Dolichos lablab perpureus L.

A proteinase inhibitor resembling Bowman-Birk family inhibitors has been purified from the seeds of cultivar HA-3 of Dolichos lablab perpureus L. The protein was apparently homogeneous as judged by SDS-PAGE, PAGE, IEF, and immunodiffusion. The inhibitor had 12 mole% 1/2-cystine and a few aromatic amino acids, and lacks tryptophan. Field bean proteinase inhibitor (FBPI) exhibited a pI of 4.3 and an M-r of 18,500 Da. CD spectral studies showed random coiled secondary structure. Conformational changes were detected in the FBPI-trypsin/chymotrypsin complexes by difference spectral studies. Apparent K-a values of complexes of inhibitor with trypsin and chymotrypsin were 2.1 x 10(7) M-1 and 3.1 X 10(7) M-1, respectively. The binary and ternary complexes of FBPI with trypsin and chymotrypsin have been isolated indicating 1:1 stoichiometry with independent sites for cognate enzymes. Amino acid modification studies showed lysine and tyrosine at the reactive sites of FBPI for trypsin and chymotrypsin, respectively.