Trypsin inhibitory activities of SdPI and its mutants.

The inhibitory activities of SdPI and its mutants on the hydrolysis of synthetic chromogenic substrates by trypsin were assayed in 100 mM Tris-HCl (pH 8.0), containing 10 mM CaCl2 at 25°C. Trypsin was pre-incubated with the inhibitor for 30 min. The reaction was initiated by addition of synthetic chromogenic substrates. Formation of p-nitroaniline was monitored continuously at 405 nm for 5 min. Inhibition constants of SdPI and mutants were determined by Lineweaver-Burk plots and further replotting of the slopes. Errors in Ki values are less than ± 10%.-, no inhibition detected.本实验于25℃、含10 mM氯化钙（CaCl₂）的100 mM三羟甲基氨基甲烷盐酸盐（Tris-HCl，pH 8.0）缓冲体系内，测定SdPI及其突变体对胰蛋白酶（trypsin）水解合成分色底物（chromogenic substrates）的抑制活性。实验前将胰蛋白酶与抑制剂预孵育30分钟，随后加入合成分色底物启动反应，于405 nm波长下连续监测对硝基苯胺（p-nitroaniline）的生成过程，监测时长为5分钟。采用莱恩韦弗-伯克（Lineweaver-Burk）作图法并对所得斜率进行二次重绘图，计算得到SdPI及其突变体的抑制常数（Ki），Ki值的误差范围小于±10%。未检测到抑制活性。