Mann K G, Williams E B, Krishnaswamy S, Church W, Giles A, Tracy R P
Department of Biochemistry, University of Vermont, Burlington 05405.
Blood. 1990 Aug 15;76(4):755-66.
This study describes a process by which serine proteases that contain an S-1 arginine subsite and active site histidine may be inactivated and subsequently quantitated using a combination of peptidyl chloromethylketone chemistry and immune recognition technology. Active site labeling and inactivation of proteases is attained by modification of the active site histidine with a peptidyl chloromethylketone. In the specific illustrations demonstrated, we used the compound biotinyl-epsilon-aminocaproyl-phenylalanylprolylarginyl chloromethylketone. This reagent reacts quantitatively and specifically with the active site histidine of a wide variety of proteases that are elaborated in the coagulation and fibrinolytic system. The inactivated enzyme(s) may be quantitated by combinations of antiprotein antibodies and avidin binding technology using the biotin moiety on the peptide inhibitor. We have demonstrated the capability of capture of inactivated enzyme products directly on to solid-phase avidin with subsequent quantitation of bound protein using specific antibodies. In the converse system we have captured specific proteases using antiprotein antibodies in the solid phase and have quantitated bound enzyme by using avidin. Subsequent detection and quantitation has been achieved using the enzymatic activity of horseradish peroxidase conjugated either to the antibody or to avidin. Both types of assays are feasible, with avidin capture being the preferred mode when enzyme is evaluated in the presence of excess zymogen, as would be common in the evaluation of most blood-clotting enzymes. Assays are illustrated for tissue plasminogen activator, plasmin, thrombin, factor Xa, and activated protein C, which can measure protease concentrations as low as 50 pmol/L. Specific applications of the assays are provided in studies of the activation of prothrombin by the prothrombinase complex and of factor X with Russell's viper venom factor X activator. These assays measure the mass of active site present in the reaction mixture and are relatively independent of subspecies of enzyme or the environment in which the activity is generated. These assay systems provide powerful tools for elucidating product-precursor relationships in multienzyme feedback reactions involving zymogen activation.
本研究描述了一种方法，通过该方法，含有S-1精氨酸亚位点和活性位点组氨酸的丝氨酸蛋白酶可被灭活，随后使用肽基氯甲基酮化学和免疫识别技术的组合进行定量。蛋白酶的活性位点标记和灭活是通过用肽基氯甲基酮修饰活性位点组氨酸来实现的。在所示的具体示例中，我们使用了化合物生物素化-ε-氨基己酰-苯丙氨酰脯氨酰精氨酰氯甲基酮。该试剂与凝血和纤溶系统中产生的多种蛋白酶的活性位点组氨酸进行定量且特异性反应。灭活的酶可以通过抗蛋白酶抗体和利用肽抑制剂上生物素部分的抗生物素蛋白结合技术的组合进行定量。我们已经证明了将灭活的酶产物直接捕获到固相抗生物素蛋白上的能力，随后使用特异性抗体对结合的蛋白质进行定量。在相反的系统中，我们使用固相中的抗蛋白酶抗体捕获特异性蛋白酶，并通过使用抗生物素蛋白对结合的酶进行定量。随后使用与抗体或抗生物素蛋白偶联的辣根过氧化物酶的酶活性实现检测和定量。两种类型的测定都是可行的，当在过量酶原存在下评估酶时，抗生物素蛋白捕获是首选模式，这在大多数凝血酶的评估中很常见。对组织纤溶酶原激活物、纤溶酶、凝血酶、因子Xa和活化蛋白C进行了测定示例，这些测定可以测量低至50 pmol/L的蛋白酶浓度。这些测定在凝血酶原酶复合物激活凝血酶原以及用罗素蝰蛇毒因子X激活剂激活因子X的研究中提供了具体应用。这些测定测量反应混合物中存在的活性位点的质量，并且相对独立于酶的亚型或产生活性的环境。这些测定系统为阐明涉及酶原激活的多酶反馈反应中的产物-前体关系提供了强大的工具。
