花生胰蛋白酶抑制剂的纯化及钝化研究

PURIFICATION AND INACTIVATION OF TRYPSIN INHIBITOR FROM PEANUT (ARACHIS HYPOGAEA L.)SEEDS

目的：从花生种子中提取纯化胰蛋白酶抑制剂，研究其聚合形式及热稳定性等性质，并采用各种巯基还原剂和蛋白酶钝化其胰蛋白酶抑制活性。方法：采用５０ｍｍｏｌ／Ｌ醋酸溶液提取，５０％～９０％丙酮分级沉淀分离及ＥＡＥＤ－ＳｅｐｈａｄｅｘＡ５０离子交换色谱从花生（ＡｒａｃｈｉｓｈｙｐｏｇａｅａＬ．）种子中提取并纯化了胰蛋白酶抑制剂（ＰＴＩ）；并以ＢＡＰＮＡ为底物测定其抑制活性、热稳定性及巯基还原剂对抑制剂的钝化作用；用ＳＤＳ－ＰＡＧＥ方法测定其分子量及蛋白酶水解敏感性。结果：ＰＴＩ是由等电点约４．３，分子量约５０００～８０００的两个多肽组成的二聚体；高度耐热、耐酸且不易被蛋白酶降解。各种巯基还原剂可不同程度地增加ＰＴＩ的热敏感性及蛋白酶水解敏感性，以二硫苏糖醇（ＤＴＩ），β－巯基乙醇（β－ＭＥ）及Ｎａ２ＳＯ３较佳。枯草杆菌蛋白酶可部分水解氧化态的ＰＴＩ，并彻底水解还原态的ＰＴＩ；而木瓜蛋白酶不能水解还原态的ＰＴＩ。结论：ＰＴＩ的热稳定性与二硫键的存在有关，还原态的ＰＴＩ增加了对热和蛋白酶水解的敏感性，巯基还原剂结合枯草杆菌蛋白酶水解可在常温下彻底钝化ＰＴＩ的活性。

Objective:The purification and inactivation of trypsin inhibitor from peanut(Archis hypogaea L.)seeds were studied. Method:The trypsin inhibitor in peanut seeds(PTI)was purified by 50 mmol/L acetic acid,50-90%(V/V) acetone fractionation,followed by passing through DEAE Sephadex A50 ion exchange column.Results:PTI consisted of two subunits with molecular weight of 5000-8000 and isoelectric point of 4.3. The purified PTI in solution was stable to heat, acids and hard to be degraded by proteases.PTI was reduced by thiols (eg.cysteine,β mercaproethanol,reduced glutathione,dithiothreitol and Na 2SO 3) and lose the ability to inhibit trypsin.Moreover,the reduced form of the inhibitor showed incresed susceptibility to heat and proteolysis by subtilisin,but the reduced form of PTI failed to be digested by papain.Conclusion:The disulfide bond plays a major role in PTI resistance to heat. The reduced form of PTI is more sensitive to heat and proteolysis by proteases.The thiols can inactivate PTI completely in conbination with subtilisin at room temperature.