萨尔瓦多型银合欢种子胰蛋白酶抑制剂的分离纯化与性质研究

Purification and characterization of a novel trypsin inhibitor from Leucaena leucocephala cv.Salvador seeds

本研究从萨尔瓦多型银合欢(Leucaena leucocephala cv.Salvador)种子中分离纯化出一种新的胰蛋白酶抑制剂,即萨尔瓦多型银合欢种子胰蛋白抑制剂(Trypsin Inhibitor from Leucaena leucocephala cv.Salvador seeds,LlsTI).成熟的种子经粉碎,生理盐水浸提之后,经CM-Sepharose阳离子交换层析得到LlsTI纯品.该胰蛋白抑制剂为19.8kDa的单体蛋白.其N末端测序结果为KGSYLRDVRG,而其内部则含有一段KTAPLVVGFLK的序列,与已知的蛋白酶抑制剂序列都不同,表明LlsTI为一种新的胰蛋白酶抑制剂,同时也含有一部分与已知Kunitz型抑制剂相保守的碱基.LlsTI对胰蛋白酶的摩尔抑制比近似为1∶1.其对于胰蛋白酶属竞争性抑制剂,其Ki值为2.77×10-10 M.LlsTI对胰蛋白酶的抑制活力表现出对高温和pH变化较高的耐受性.但其活性受100mM DTT影响.本研究对LlsTI对其它丝氨酸类蛋白酶的抑制作用也做了测定,其中只有胰蛋白酶的活性受到了抑制.根据圆二色谱的分析结果,LlsTI是一种αβ蛋白,其二级结构在室温下的水和缓冲液Tris-HCl(pH7.5)中组成相似.

A novel trypsin inhibitor named LlsTI was purified from the seeds of Leucaena leucocephala cv. Salvador. It is a monomeric protein with a molecular weight of 19.8 kDa. The N-terminal of LlsTI was determined to be KGSYLRDVRG, and the internal sequencing was determined as KTAPLVVG- FLK, showing no similarities to any of the known trypsin inhibitors. LlsTI is a competitive inhibitor to wards trypsin with the Ki value of 2.77 X 10-1 M. The trypsin-inhibitory activity of LlsTI appears high stability to the changes of temperature and pH, but can be impacted by DTT at a concentration of 100mM. The inhibitory effects of LlsTI toward other serine proteases were assayed, and only the trypsin activity can be blocked. According to circular dichroism spectroscopy, LlsTI displays a pattern and its secondary structure has no marked changes in water or Tris-HCl buffer （pH 7.5） at room temperature.