Isolation and Characterization of Four Isoinhibitors from Cowpea (Vigna unguiculata (L.) Walp.) Seeds

Four isoinhibitors of trypsin have been isolated from the cowpea, Vigna unguiculata (L.) Walp., in a highly purified state, as indicated by Urea-SDS-polyacrylamide gel electrophoresis, equilibrium chromatography, and amino acid analysis. The amino acid composition was characterized by the absence of free -SH groups, high half-cysteine, and the absence of tryptophan and methionine in isoinhibitors II and III. Isoinhibitor II contained 96 amino acid residues, whereas isoinhibitor III contained 80. Isoinhibitor II had lysine in the reactive site, while isoinhibitor III had arginine. Isoinhibitor IA inhibited trypsin only, while isoinhibitors IB, II, and III inhibited both trypsin and α-chymotrypsin, indicating that each one had an independent site for each enzyme (trypsin and chymotrypsin). The amino acid sequence of isoinhibitor III exhibited a close homology with Macrotyloma axillare DE3 (81%), lima bean IV (78%), Macrotyloma axillare DE4 (72%), and mung bean (65%) isoinhibitors.