Specificity assay of serine proteinases by reverse-phase high-performance liquid chromatography analysis of competing oligopeptide substrate library
Specificity assay of serine proteinases by reverse-phase high-performance liquid chromatography analysis of competing oligopeptide substrate library
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DOI：
10.1006/abio.2000.4886
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发表时间：
2001-01-15
影响因子：
2.9
通讯作者：
Gráf, L
中科院分区：
文献类型：
--
作者：
Antal, J;Pál, G;Gráf, L
本文提出了一种高效液相色谱法，用于丝氨酸蛋白酶活性和特异性的快速分析。该方法应用了精心设计的肽库，其中各个组分仅在酶的潜在切割位点处不同。该库有七个成员代表七个不同的切割位点，它提供了胰蛋白酶和胰凝乳蛋白酶样酶的底物。单个肽底物在酶促反应期间竞争蛋白酶。通过组分的RP-HPLC分离监测反应。我们描述了竞争性肽底物库的系统设计和系统的测试与八种不同的丝氨酸蛋白酶。通过新方法确定的研究酶的特异性概况与文献中报道的基本相同，验证了系统表征底物特异性的能力。测试还表明，该系统甚至可以检测到酶的两种异构体的细微特异性差异。除了记录定性特异性概况外，还可以对系统提供的数据进行定量分析，产生特异性常数值。这种方法可以是一个有用的工具，用于快速分析未知的基因产物以及蛋白质工程产生的新形式的酶。(C)北京：科学出版社.
In this paper we present an HPLC method developed for quick activity and specificity analysis of serine proteinases. The method applies a carefully designed peptide library in which the individual components differ only at the potential cleavage site for enzymes. The library has seven members representing seven different cleavage sites and it offers substrates for both trypsin and chymotrypsin-like enzymes. The individual peptide substrates compete for the proteinase during the enzymatic reaction. The reaction is monitored by RP-HPLC separation of the components. We describe the systematic design of the competitive peptide substrate library and the test of the system with eight different serine proteinases. The specificity profiles of the investigated enzymes as determined by the new method were essentially identical to the ones reported in the literature, verifying the ability of the system to characterize substrate specificity. The tests also demonstrated that the system could detect even subtle specificity differences of two isoforms of an enzyme. In addition to recording qualitative specificity profiles, data provided by the system can be analyzed quantitatively, yielding specificity constant values. This method can be a useful tool for quick analysis of uncharacterized gene products as well as new forms of enzymes generated by protein engineering. (C) 2001 Academic Press.