A standard operating procedure for an enzymatic activity inhibition assay


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.

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Authors acknowledge the COST project ARBRE-MOBIEU CA15126.

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Correspondence to Daumantas Matulis.

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Special Issue: COST Action CA15126, MOBIEU: Between atom and cell.

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Smirnovienė, J., Baranauskienė, L., Zubrienė, A. et al. A standard operating procedure for an enzymatic activity inhibition assay. Eur Biophys J 50, 345–352 (2021). https://doi.org/10.1007/s00249-021-01530-8

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  • Enzyme inhibition
  • Tight binding
  • Hill equation
  • Morrison equation