Sumathi S, Pattabiraman T N
Biochim Biophys Acta. 1977 Nov 23;485(1):167-78. doi: 10.1016/0005-2744(77)90204-2.
A trypsin/chymotrypsin inhibitor was isolated from the tubers of Alocasia macrorhiza by extraction at pH 7.6, heat treatment at 80 degrees C, ammonium sulphate precipitation and successive column chromatography on CM-cellulose, DEAE-Sephadex A-50 and Sephadex G-100. The inhibitor was pure by cellulose acetate electrophoresis. The molecular weight was approximately 32 000 as determined by gel filtration on Sephadex G-100. The inhibitor acted on bovine trypsin, human trypsin and bovine chymotrypsin. It had no action on human chymotrypsin, subtilisin BPN', pronase, Aspergillus oryzae protease, human and porcine pepsins. The binding sites for bovine trypsin and chymotrypsin are not mutually exclusive. The inhibitor was stable over a pH range of 1--10. The purified inhibitor was far more thermostable than the crude inhibitor. The purified inhibitor lost only 33% of activity on heat treatment at 95 degrees C for 2 h. Trinitrobenzene sulphonate treatment resulted in the loss of antichymotryptic activity faster than the antitrypsin activity of the inhibitor.
通过在pH 7.6下提取、80℃热处理、硫酸铵沉淀以及先后在CM - 纤维素、DEAE - 葡聚糖A - 50和葡聚糖G - 100柱上进行层析，从海芋块茎中分离出一种胰蛋白酶/糜蛋白酶抑制剂。经醋酸纤维素电泳检测，该抑制剂呈纯态。通过在葡聚糖G - 100上进行凝胶过滤测定，其分子量约为32000。该抑制剂作用于牛胰蛋白酶、人胰蛋白酶和牛糜蛋白酶。对人糜蛋白酶、枯草杆菌蛋白酶BPN'、链霉蛋白酶、米曲霉蛋白酶、人胃蛋白酶和猪胃蛋白酶均无作用。牛胰蛋白酶和糜蛋白酶的结合位点并非相互排斥。该抑制剂在pH 1 - 10范围内稳定。纯化后的抑制剂比粗提抑制剂热稳定性高得多。纯化后的抑制剂在95℃热处理2小时后仅损失33%的活性。三硝基苯磺酸盐处理导致抑制剂抗糜蛋白酶活性的丧失比抗胰蛋白酶活性更快。
