Maliopoulou T B, Dionyssiou-Asteriou A, Loucopoulos D
J Biochem Biophys Methods. 1980 Jun;2(6):357-60. doi: 10.1016/s0165-022x(80)90052-4.
A method is described for the assay of proteolytic activity, based on the digestion of L-[4,5-3H]leucine globin. L-[4,5-3H]Leucine was incorporated into the substrate at the stage of haemoglobin biosynthesis, using rabbit erythrocytes. Assay methods for proteolytic enzymes have been based on the digestion of haemoglobin, serum albumin or casein, and the determination of the trichloroacetic acid-soluble products [1,2]. More sensitive methods have been developed by using haemoglobin labelled with a fluorescent [3-5] or radioactive marker [6,7]. These methods avoid the errors which beset the Anson procedure, such as interference by impurities (purines at 280 nm and reducing compounds at 700 nm) [8]. However, methods using labelled proteins as a substrate present a number of problems, the most troublesome of which are the high blank values and the use of non-physiological substrates when chemically modified proteins are employed. In the present communication a simple and sensitive method for the assay of proteolytic enzyme activity is described. This is based on the digestion of L-[4,5-3H]leucine globin by proteolytic enzymes and radioactivity measurement of the trichloroacetic acid soluble cleavage products.
本文描述了一种基于L-[4,5-³H]亮氨酸标记的球蛋白消化来测定蛋白水解活性的方法。在血红蛋白生物合成阶段，利用兔红细胞将L-[4,5-³H]亮氨酸掺入底物中。蛋白水解酶的测定方法一直基于血红蛋白、血清白蛋白或酪蛋白的消化以及对三氯乙酸可溶性产物的测定[1,2]。通过使用荧光标记[3-5]或放射性标记[6,7]的血红蛋白，已开发出更灵敏的方法。这些方法避免了困扰安森（Anson）法的误差，如杂质干扰（280nm处的嘌呤和700nm处的还原化合物）[8]。然而，使用标记蛋白质作为底物的方法存在许多问题，其中最麻烦的是高空白值以及使用化学修饰蛋白质时使用非生理性底物。在本报告中，描述了一种简单且灵敏的测定蛋白水解酶活性的方法。该方法基于蛋白水解酶对L-[4,5-³H]亮氨酸标记的球蛋白的消化以及对三氯乙酸可溶性裂解产物的放射性测量。
