Identification of Kunitz inhibitor from Cassia obtusifolia L. and its inhibitory effect against Pieris rapae proteases
大决明Kunitz抑制剂的鉴定及其对菜青虫中肠蛋白酶的抑制作用

A trypsin inhibitor from Cassia obtusifolia seeds,a well known Chinese herb,was isolated to apparent homogeneity by a combination of distilled water extraction,ammonium sulfate precipitation,Sepharose 4B-trypsin affinity and Sephadex G-75 chromatography.SDS-PAGE and MALDI-TOF analyses show that this inhibitor consisted of a single polypeptide chain with accurate molecular mass of 19812.55 Da.The inhibitor contained large quantities of isoleucine,valine and phenylalanine.Peptide mass fingerprint of the inhibitor showed eight signal clusters.The inhibitor had one reactive site involved with lysine residue.Simulated gastric fluid digestion and fluorescence spectra show disulfide linkage and lysine residue were important in maintaining the biologically active conformation of this inhibitor.This inhibitor lost inhibitory activity and resistance to pepsin under reductive and lysine-modified process.Partial amino acid sequence of the purified protein from MS/MS showed a high degree of homology with various members of the Kunitz-inhibitor family.Moreover,trypsin-like activity in midgut of Pieris rapae larvae was substantially inhibited by the purified inhibitor,which showed equivalent inhibitory activity with soybean Bowman-Birk inhibitor.