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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.
One trypsin inhibitor was obtained from the traditional medicinal plant Cassia torai using deionized water extraction, thermal denaturation, ammonium sulfate salting-out, affinity chromatography and molecular sieve chromatography.DS-PAGE and MALDI- TOF was detected as a single polypeptide chain with an accurate molecular weight of 19812.55 Da. Amino acid composition analysis showed that the inhibitor contained the highest content of isoleucine, valine and phenylalanine, and there were 8 major signal clusters in peptide fingerprinting Simulated gastric juice experiments and fluorescence spectroscopy results show that disulfide bonds and lysine residues are essential for maintaining the native active conformation of the inhibitor.When the inhibitor is reduced or the lysine residue is modified, it inhibits Activity and resistance to pepsin.A partial amino acid sequence of the inhibitor was determined by tandem mass spectrometry (MS / MS) and compared with a number of Kunitz protease inhibitors of leguminosae and found that there was a significant difference High homology.At the same time, the inhibitor can inhibit intestinal trypsin activity of cabbage worm, its inhibitory effect and soybean Bowman-Birk protease inhibitors inhibitory effect.