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. 2022 Oct 5;12(19):e4528.
doi: 10.21769/BioProtoc.4528.

Assay for Protealysin-like Protease Inhibitor Activity

Igor M Berdyshev  1 , Maria A Karaseva  1 , And Ilya V Demidyuk  1
Affiliations

Assay for Protealysin-like Protease Inhibitor Activity

Igor M Berdyshev et al. Bio Protoc. .

Abstract

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.

Keywords: Emfourin; Internally quenched fluorescent peptide substrate; Metalloprotease activity; Protealysin; Proteinaceous protease inhibitor.

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Conflict of interest statement

Competing interests The authors declare no conflict of interest.

Figures

Figure 1.
Figure 1.. Emfourin-mediated inhibition of the Abz-RSVIK(Dnp) hydrolysis catalyzed by protealysin.
The Abz-RSVIK(Dnp) concentrations are 30 (A) and 90 (B) μM; the emfourin concentrations are 0, 0.5, 1, 1.5, 2, 2.5, and 3.5 nM; the protealysin concentration is 50 pM. The reactions were performed at 37 °C. Abz-RS (2-aminobenzoyl-L-arginyl-L-serin) is the product of the substrate hydrolysis. Conversion of the relative fluorescence units into the product concentration was made using calibration curves, as described previously ( Karaseva et al., 2019 ).
Figure 2.
Figure 2.. Kinetics of Abz-RSVIK(Dnp) hydrolysis by protealysin at different concentrations.
The protealysin concentrations are 0, 25, 50, 100, 250, 500, and 2,500 pM; the Abz-RSVIK(Dnp) concentration is 30 μM. The reactions were performed at 37 °C. Abz-RS (2-aminobenzoyl-L-arginyl-L-serin) is the product of the substrate hydrolysis. Conversion of the relative fluorescence units into the product concentration was made using calibration curves, as described previously ( Karaseva et al., 2019 ).
Figure 3.
Figure 3.. Effect of DMSO on Abz-RSVIK(Dnp) hydrolysis rate by protealysin.
The Abz-RSVIK(Dnp) concentration is 16 μM; the protealysin concentration is 50 pM. The reactions were performed at 37 °C.
Figure 4.
Figure 4.. Michaelis–Menten saturation curve showing the relation between Abz-RSVIK(Dnp) concentration and the protealysin catalyzed reaction rate.
The protealysin concentration is 50 pM. The reactions were performed at 37 °C.
None
See this image and copyright information in PMC

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