Berdyshev Igor M, Karaseva Maria A, Demidyuk And Ilya V
Institute of Molecular Genetics of National Research Centre "Kurchatov Institute," Moscow, Russia.
Bio Protoc. 2022 Oct 5;12(19). doi: 10.21769/BioProtoc.4528.
Here, we present the first quantitative method for the activity analysis of protealysin-like protease (PLP) inhibitors. This approach is based on a previously developed method for protealysin activity determination by hydrolysis of internally quenched fluorescent peptide substrate 2-aminobenzoyl-L-arginyl-L-seryl-L-valyl-L-isoleucyl-L-(ε-2,4-dinitrophenyl)lysine. In this protocol, we significantly reduced enzyme concentration and introduced some minor modifications to decrease variation between replicates. The protocol was validated using emfourin, a novel proteinaceous metalloprotease inhibitor. Data obtained demonstrates that the developed assay method is an affordable approach for characterizing and screening various PLP inhibitors. Graphical abstract.
在此，我们提出了第一种用于类蛋白水解酶样蛋白酶（PLP）抑制剂活性分析的定量方法。该方法基于先前开发的一种通过水解内部淬灭的荧光肽底物2-氨基苯甲酰-L-精氨酰-L-丝氨酰-L-缬氨酰-L-异亮氨酰-L-（ε-2,4-二硝基苯基）赖氨酸来测定蛋白水解酶活性的方法。在本方案中，我们显著降低了酶浓度，并进行了一些小的修改以减少重复实验之间的差异。该方案使用新型蛋白质金属蛋白酶抑制剂emfourin进行了验证。所获得的数据表明，所开发的检测方法是一种用于表征和筛选各种PLP抑制剂的经济实惠的方法。图形摘要。
