NEW ASSAY FOR CATHEPSIN B1 AND OTHER THIOL PROTEINASES
NEW ASSAY FOR CATHEPSIN B1 AND OTHER THIOL PROTEINASES
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DOI：
10.1016/0003-2697(72)90302-8
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发表时间：
1972-01-01
影响因子：
2.9
通讯作者：
BARRETT, AJ
中科院分区：
文献类型：
--
作者：
BARRETT, AJ
描述了一种新的组织蛋白酶B1测定方法。底物为α- n -苯甲酰-dl-精氨酸2-萘酰胺，释放游离2-萘胺后与Fast石榴石GBC偶氮偶联反应。巯基蛋白酶需要半胱氨酸的激活，而半胱氨酸通常会干扰偶联反应，但通过用4-氯甲苯甲酸酯阻断巯基试剂可以克服这一困难。有颜色的反应产物的量与酶的浓度在很宽的范围内呈线性关系。由于萘酰胺的水解率高，着色产物的摩尔消光率高，该测定法的灵敏度比以前使用的底物为4-硝基苯胺的测定法高35倍。灵敏度的增加允许使用足够短的孵育时间，以尽量减少过去在测定条件下由于酶活性的逐渐丧失而可能发生的错误。有色反应产物非常稳定，可提取为正丁醇，有利于浑浊样品的测定。该方法也可直接应用于木瓜蛋白酶的测定，并且用另一种底物替代，可能对组织蛋白酶C有价值。
A new assay procedure for cathepsin B1 is described. The substrate is α-N-benzoyl-dl-arginine 2-naphthylamide, and the release of free 2-naphthylamine is followed by an azo coupling reaction with Fast Garnet GBC. The thiol proteinase requires activation by cysteine, which would normally interfere in the coupling reaction, but the difficulty is overcome by blocking the thiol reagent with 4-chloromercuribenzoate. The amount of the colored reaction product is linearly related to enzyme concentration over a wide range. Because of the high rate of hydrolysis of the naphthylamide, and the high molar extinction of the colored product, the assay is about 35 times more sensitive than that previously used, in which the substrate was the 4-nitroanilide. The increase in sensitivity permits the use of incubation times short enough to minimize errors that may have occurred in the past due to the progressive loss in activity of the enzyme under the conditions of the assay. The colored reaction product is very stable, and is extractable into n-butanol, which facilitates the assay of turbid samples. The procedure is directly applicable to the assay of papain, also, and with substitution of another substrate, would probably be valuable for cathepsin C.