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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. | Turk J Biol 30 (2006) 207-215 © TÜB‹TAK Isolation and Characterization of Four Isoinhibitors from Cowpea (Vigna unguiculata (L.) Walp.) Seeds Reda Helmy Ahmed SAMMOUR Department of Botany, Faculty of Science, Tanta University - EGYPT Received: 12.10.2005 Abstract: 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. Key Words: CPTI, cowpea trypsin inhibitor, N-α-benzoyl-L-arginine-p-nitroanilide (BAPNA), P-nitrophenyl-P-guanidinobenzoate HCl (NPGB), N-benzoyl-L-tyrosine-ethyl ester (BTEE) Introduction Enzyme inhibitors found in seeds have received particular attention because of their potentially deleterious effects on animal and human nutrition, and their possible role in the defense of plants against microbial and insect pests. Three functions have been proposed for plant proteinase inhibitors: as regulatory agents in controlling endogenous proteinases, as storage proteins, and as protective agents directed against insects or microbial proteinases (1-4). The protein inhibitors of trypsin are widespread in the plant kingdom, with the .