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(英文)

Identification of Kunitz inhibitor from Cassia obtusifolia L. and its inhibitory effect against Pieris rapae proteases

采用去离子水抽提、热变性、硫酸铵盐析及亲和层析和分子筛层析等方法,从传统药用植物大决明种子中得到1种胰蛋白酶抑制剂.SDS-PAGE和MALDI-TOF检测为单一多肽链,其精确分子量为19812.55 Da.氨基酸组成分析表明,该抑制剂中异亮氨酸、缬氨酸和苯丙氨酸含量最高.肽指纹图谱中有8个主要的信号簇.模拟胃液实验和荧光光谱结果表明,二硫键和赖氨酸残基对于维持该抑制剂的天然活性构象必不可少.当该抑制剂被还原或赖氨酸残基被修饰后,其抑制活性及对胃蛋白酶的抗性会有较大程度的丧失.通过串联质谱(MS/MS)测定该抑制剂的部分氨基酸序列,并与多种豆科Kunitz蛋白酶抑制剂进行比对,发现有较高的同源性.同时,该抑制剂能够抑制菜青虫中肠类胰蛋白酶的活力,其抑制效果与大豆Bowman-Birk蛋白酶抑制剂的抑制效果相当.

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 MAI.DI-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.