Jongsma M A, Bakker P L, Stiekema W J
Department of Molecular Biology, DLO-Centre for Plant Breeding and Reproduction Research (CPRO-DLO), Wageningen, The Netherlands.
Anal Biochem. 1993 Jul;212(1):79-84. doi: 10.1006/abio.1993.1294.
An improved, time efficient, visual assay for quantitative determination of proteinase inhibitor activity in protein extracts is reported. Proteinase inhibitor activity of mammalian, bacterial, and fungal serine proteinases can be quantified. The method relies on radial diffusion of proteinase inhibitor containing extracts from a central well through an agar gel containing a serine proteinase. After an incubation period the agar gel is stained via the diazo coupling of the beta-naphthol produced by the enzymatic hydrolysis of N-acetyl-DL-phenylalanine-beta-naphthyl-ester. Circular zones containing inhibitor-proteinase complexes remain colorless while the region containing only proteinase shows a bright pink-purple color. A reference curve relates the diameter of the colorless zone to the logarithm of the proteinase inhibitor concentration. The error in the estimation of a proteinase inhibitor quantity varying between 10 and 1000 pmol is 4-12%. The sensitivity of the assay is approximately 2-20 pmol of inhibited proteinase molecules depending on the inhibitor-proteinase complex assayed. The sensitivity of the assay can be enhanced 10-fold or more by dilution of the proteinase concentration in the agar and by a reduction of the agar thickness.
报道了一种改进的、省时的视觉检测方法，用于定量测定蛋白质提取物中的蛋白酶抑制剂活性。可对哺乳动物、细菌和真菌丝氨酸蛋白酶的蛋白酶抑制剂活性进行定量。该方法依赖于含蛋白酶抑制剂的提取物从中心孔通过含有丝氨酸蛋白酶的琼脂凝胶进行径向扩散。孵育一段时间后，通过对N-乙酰-DL-苯丙氨酸-β-萘酯酶促水解产生的β-萘酚进行重氮偶联，对琼脂凝胶进行染色。含有抑制剂-蛋白酶复合物的圆形区域保持无色，而仅含蛋白酶的区域呈现亮粉紫色。参考曲线将无色区域的直径与蛋白酶抑制剂浓度的对数相关联。在估计10至1000皮摩尔之间变化的蛋白酶抑制剂数量时，误差为4-12%。根据所检测的抑制剂-蛋白酶复合物，该检测方法的灵敏度约为2至20皮摩尔被抑制的蛋白酶分子。通过稀释琼脂中的蛋白酶浓度和减小琼脂厚度，可将检测方法的灵敏度提高10倍或更多。
