Plant Protease Inhibitors from Abelmoschus moschatus L. Seeds: Characterization, Inhibitory and Kinetic Studies

Abstract

Protease inhibitors have been isolated and reported in many plant species and mono-headed trypsin inhibitors are the most common type. The objective of the present study was to characterize mono-headed kunitz type trypsin inhibitors, AMTI-I and AMTI-II isolated from the seeds of Abelmoschus moschatus with respect to their specificity, mode of action and kinetic studies. Mono-headed inhibitors were isolated and purified following conventional methods of protein purification. Inhibitory activities of mono-headed inhibitors against various proteases of mammalian, bacterial and fungal origin has been studied. AMTI-I and AMTI-II were discovered to be serpins, with a high affinity for trypsin and a moderate affinity for porcine elastase, Staphylococcus aureus protease, and Aspergillus oryzae protease. AMTI-I and AMTI-II have shown non-competitive type of inhibition towards 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 findings indicate that trypsin forms a stable 1:1 complex with both AMTI-I and AMTI-II. Chemical modification of the inhibitors' functional groups by selective reagents revealed that arginine residues are required for their trypsin inhibitory activities. Studies on the specificity of protease inhibitors are critical for understanding their physiological role, as well as the control mechanisms involved in the regulation of proteolysis in biological systems.