Jean F, Basak A, Chrétien M, Lazure C
J. A. De Sève Laboratory of Molecular Neuroendocrinology, Clinical Research Institute of Montréal (Affiliated with l'Université de Montréal), Québec, Canada.
Anal Biochem. 1991 May 1;194(2):399-406. doi: 10.1016/0003-2697(91)90248-r.
A radiometric procedure to detect the presence of proteolytic enzymes and analyze their substrate specificity is described. The enzymatic activity is first measured by the release into solution of a radiolabeled reporter group from an immobilized peptidyl substrate. Two peptidyl substrates encompassing multiple cleavage sites, a derivative of Leu-enkephalin and a peptide related to the bait region of human alpha 2-macroglobulin, are prepared and linked via a spacer molecule to an insoluble support. The labeled peptides released are then separated by high-performance liquid chromatography. The position of the released peptides upon chromatography allows direct identification of the sites of cleavage. The assay, using a radioactive iodinated tyrosine residue as reporter group, is extremely sensitive (less than 0.02 pg/ml of trypsin), reproducible, and easy to perform while yielding unambiguous identification of the sites of cleavage. This assay can be used to detect the presence of enzymatic activities and/or of enzyme inhibitors. Furthermore, it can be easily adapted to detect from a variety of sources all four classes of enzymes known by using appropriate peptidyl substrate sequences, buffer, pH, and incubation conditions.
本文描述了一种用于检测蛋白水解酶的存在并分析其底物特异性的放射性测定方法。酶活性首先通过将固定化肽基底物中的放射性标记报告基团释放到溶液中来测量。制备了两种包含多个切割位点的肽基底物，亮氨酸脑啡肽的衍生物和与人α2-巨球蛋白诱饵区域相关的肽，并通过间隔分子连接到不溶性支持物上。然后通过高效液相色谱法分离释放的标记肽。色谱分离后释放肽的位置可直接鉴定切割位点。该测定法使用放射性碘化酪氨酸残基作为报告基团，极其灵敏（小于0.02 pg/ml胰蛋白酶），可重复且易于操作，同时能明确鉴定切割位点。该测定法可用于检测酶活性和/或酶抑制剂的存在。此外，通过使用适当的肽基底物序列、缓冲液、pH和孵育条件，它可以很容易地适用于从各种来源检测所有四类已知的酶。
