Four trypsin inhibitors, AMTI-I, AMTI-II, AMTI-III, and AMTI-IV, have been isolated and purified to homogeneity from the seeds of Abelmoschus moschatus following ammonium sulphate fractionation, DEAE-cellulose ion exchange chromatography and gel permeation on Sephadex G-100, and their molecular weights were determined to be 22.4, 21.2, 20.8 and 20.2 kDa respectively by SDS-PAGE. While all the four inhibitors were very active against bovine trypsin, two of them (AMTI-III and AMTI-IV) showed moderate activity towards bovine chymotrypsin. AMTI-I and AMTI-II were found to be glycoproteins with neutral sugar content of 2.8 and 4 %, respectively, and all the four inhibitors were devoid of free sulphhydryl groups. The inhibitors were quite stable up to 80 °C for 10 min and were not affected at alkaline as well as acidic conditions tested. Treating them with 8 M urea and 1 % SDS for 24 h at room temperature did not result in any loss of their antitryptic activities. However, they lost considerable antitryptic activity when treated with 6 M guanidine hydrochloride. Activities of the inhibitors were unaffected even after their reduction with DTT suggesting that disulphide bonds are not needed for their inhibitory activities.

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Objective:The objective of the present study was to characterize the monoheaded trypsin inhibitors, Abelmoschus moschatus trypsin inhibitor-I (AMTI-I) and AMTI-II from the seeds of A. moschatus with respect to their specificity, mode of action, and active site residues.Methods: Standard methods were followed in determining inhibitory activities of monoheaded inhibitors. IC 50 values and inhibitory constants (Ki) of AMTI-I and AMTI-II were determined. Studies on complex formation and chemical modification of inhibitors were performed.Results: AMTI-I and AMTI-II were found to be serpins, strongly active against trypsin, moderately active against porcine elastase, Staphylococcus aureus protease, and Aspergillus oryzae protease. AMTI-I and AMTI-II have shown non-competitive type of inhibition toward bovine trypsin with K i values of inhibitors for trypsin found to be 0.25±0.02 nM and 0.22±0.06 nM, respectively. Complex studies revealed the formation of stable 1:1 complex of trypsin with both AMTI-I and AMTI-II. Chemical modification of the functional groups of the inhibitors by selective reagents indicated that arginine residues are essential for their trypsin inhibitory activities. Conclusion:Investigations on the specificity of protease inhibitors are important for understanding their physiological role, control mechanisms involved in the regulation of proteolysis in biological systems and mode of action.

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