Niinobe M, Hitomi Y, Fujii S
J Biochem. 1980 Mar;87(3):779-83. doi: 10.1093/oxfordjournals.jbchem.a132807.
A convenient and highly sensitive colorimetric assay for various proteases, such as trypsin, chymotrypsin, plasmin, thrombin, and urokinase is described. The substrates used are alpha-naphthyl ester derivatives of N alpha-tosyl-L-lysine, N alpha-acetylglycyl-L-ination of alpha-naphthol released from them. Use of these alpha-naphthyl ester derivatives made the method more sensitive than the use of the corresponding methyl or ethyl ester derivatives. The minimum detectable concentrations of trypsin, chymotrypsin, plasmin, thrombin and urokinase in this method were about 0.002 micrograms, 0.01 microgram, 0.002 CU, 0.01 IU, and 2 IU, respectively. The Km values of trypsin and thrombin for TLNE were 0.11 mM and 0.15 mM while those for TLME were 2.5 mM and 6.7 mM, respectively; the Km values of chymotrypsin for ATNE and ATEE were 0.18 mM and 0.7 mM, respectively; and the Km values of urokinase for AGLNE and AGLME were 0.17 mM and 4 mM, respectively. Zymograms of various proteases were easy to prepare using these alpha-naphthyl ester substrates, and zymograms of trypsin and chymotrypsin were made with TLNE and ATNE, respectively, as substrates.
本文描述了一种便捷且高灵敏度的比色法，可用于检测多种蛋白酶，如胰蛋白酶、胰凝乳蛋白酶、纤溶酶、凝血酶和尿激酶。所用底物是Nα-甲苯磺酰-L-赖氨酸、Nα-乙酰甘氨酰-L-的α-萘酯衍生物，通过检测从这些底物释放的α-萘酚的显色反应来测定蛋白酶活性。使用这些α-萘酯衍生物使得该方法比使用相应的甲酯或乙酯衍生物更灵敏。此方法中胰蛋白酶、胰凝乳蛋白酶、纤溶酶、凝血酶和尿激酶的最低可检测浓度分别约为0.002微克、0.01微克、0.002 CU、0.01 IU和2 IU。胰蛋白酶和凝血酶对甲苯磺酰-L-赖氨酸-α-萘酯（TLNE）的Km值分别为0.11 mM和0.15 mM，而对甲苯磺酰-L-赖氨酸甲酯（TLME）的Km值分别为2.5 mM和6.7 mM；胰凝乳蛋白酶对乙酰-L-酪氨酸-α-萘酯（ATNE）和乙酰-L-酪氨酸乙酯（ATEE）的Km值分别为0.18 mM和0.7 mM；尿激酶对N-乙酰甘氨酰-L-酪氨酸-α-萘酯（AGLNE）和N-乙酰甘氨酰-L-酪氨酸甲酯（AGLME）的Km值分别为0.17 mM和4 mM。使用这些α-萘酯底物很容易制备各种蛋白酶的酶谱，分别以TLNE和ATNE为底物制备了胰蛋白酶和胰凝乳蛋白酶的酶谱。
