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. 2021 May;50(3-4):345-352.
doi: 10.1007/s00249-021-01530-8. Epub 2021 Apr 16.

A standard operating procedure for an enzymatic activity inhibition assay

Joana Smirnovienė  1 , Lina Baranauskienė  1 , Asta Zubrienė  1 , Daumantas Matulis  2
Affiliations

A standard operating procedure for an enzymatic activity inhibition assay

Joana Smirnovienė et al. Eur Biophys J. 2021 May.

Abstract

This Standard Operating Protocol (SOP) describes the key steps of experimental setup for an inhibition assay of enzymatic activity. The protocol begins with the design of an experiment, including the choice of a catalytic reaction, optimal conditions, fraction and concentration of the active enzyme, substrate and inhibitor concentrations and the positive and negative controls. The protocol ends with the data analysis followed by a typical example of an experiment. Despite an apparently standard procedure, the assay has a number of possible pitfalls such as low fraction of the active enzyme or errors in the analysis such as application of an improper model or incorrect determination of the inhibition constant while not recognizing the dependence on enzyme concentration. The protocol provides examples of necessary steps and controls to avoid these problems and obtain highly reliable results.

Keywords: Enzyme inhibition; Hill equation; Morrison equation; Tight binding.

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References

    1. Bisswanger H (2014) Enzyme assays. Perspect Sci 1:41–55. https://doi.org/10.1016/j.pisc.2014.02.005 - DOI
    1. Copeland RA (2005) Evaluation of enzyme inhibitors in drug discovery: a guide for medicinal chemists and pharmacologists, Methods of biochemical analysis. Wiley-Interscience, Hoboken, NJ
    1. Copeland RA (2013) Evaluation of enzyme inhibitors in drug discovery a guide for medicinal chemists and pharmacologists. John Wiley & Sons, Inc
    1. Dudutienė V, Zubrienė A, Smirnov A, Gylytė J, Timm D, Manakova E, Gražulis S, Matulis D (2013) 4-Substituted-2,3,5,6-tetrafluorobenzenesulfonamides as inhibitors of carbonic anhydrases I, II, VII, XII, and XIII. Bioorg Med Chem 21:2093–2106. https://doi.org/10.1016/j.bmc.2013.01.008 - DOI - PubMed
    1. Fink AL, Meehan P (1979) Detection and accumulation of tetrahedral intermediates in elastase catalysis. Proc Natl Acad Sci U S A 76:1566–1569. https://doi.org/10.1073/pnas.76.4.1566 - DOI - PubMed - PMC

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