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.

Graphic abstract

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2


  1. Bisswanger H (2014) Enzyme assays. Perspect Sci 1:41–55. https://doi.org/10.1016/j.pisc.2014.02.005

    Article  Google Scholar 

  2. 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

    Google Scholar 

  3. Copeland RA (2013) Evaluation of enzyme inhibitors in drug discovery a guide for medicinal chemists and pharmacologists. John Wiley & Sons, Inc

  4. 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

    CAS  Article  PubMed  Google Scholar 

  5. 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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. Greis KD (2007) Mass spectrometry for enzyme assays and inhibitor screening: an emerging application in pharmaceutical research. Mass Spectrom Rev 26:324–339. https://doi.org/10.1002/mas.20127

    CAS  Article  PubMed  Google Scholar 

  7. Holdgate GA, Meek TD, Grimley RL (2018) Mechanistic enzymology in drug discovery: a fresh perspective. Nat Rev Drug Discov 17:115–132. https://doi.org/10.1038/nrd.2017.219

    CAS  Article  PubMed  Google Scholar 

  8. Jogaitė V, Zubrienė A, Michailovienė V, Gylytė J, Morkūnaitė V, Matulis D (2013) Characterization of human carbonic anhydrase XII stability and inhibitor binding. Bioorg Med Chem 21:1431–1436. https://doi.org/10.1016/j.bmc.2012.10.016

    CAS  Article  PubMed  Google Scholar 

  9. Kowang TO, Long CS (2015) Design of experiment—an integration of Fisher, Taguchi and Shainin DOE methodology [WWW Document]. Appl Mech Mater. https://doi.org/10.4028/www.scientific.net/AMM.789-790.1201

  10. Kuzmič P (2011) Optimal design for the dose–response screening of tight-binding enzyme inhibitors. Anal Biochem 419:117–122. https://doi.org/10.1016/j.ab.2011.06.013

    CAS  Article  PubMed  Google Scholar 

  11. Kuzmič P (2015) History, variants and usage of the “Morrison equation” in enzyme inhibition kinetics, BioKin Technical Note TN-2015-01, Biokin Ltd., Watertown, MA [Online]. http://www.biokin.com/TN/2015/01

  12. Kuzmič P (2020) Optimal duration of the preincubation phase in enzyme inhibition experiments. https://doi.org/10.26434/chemrxiv.12016974.v1

  13. Kuzmič P, Elrod KC, Cregar LM, Sideris S, Rai R, Janc JW (2000) High-throughput screening of enzyme inhibitors: simultaneous determination of tight-binding inhibition constants and enzyme concentration. Anal Biochem 286:45–50. https://doi.org/10.1006/abio.2000.4685

    CAS  Article  PubMed  Google Scholar 

  14. Liu M-S, Gong S, Yu H-H, Taylor DW, Johnson KA (2019) Chapter Thirteen—Kinetic characterization of Cas9 enzymes, In: Bailey, S. (Ed.), Methods in enzymology, CRISPR-Cas Enzymes. Academic Press, pp 289–311. https://doi.org/10.1016/bs.mie.2018.10.022

  15. Morrison JF (1969) Kinetics of the reversible inhibition of enzyme-catalysed reactions by tight-binding inhibitors. Biochim Biophys Acta 185:269–286. https://doi.org/10.1016/0005-2744(69)90420-3

    CAS  Article  PubMed  Google Scholar 

  16. Onyeogaziri FC, Papaneophytou C (2019) A general guide for the optimization of enzyme assay conditions using the design of experiments approach. SLAS Discov Adv Life Sci R D 24:587–596. https://doi.org/10.1177/2472555219830084

    CAS  Article  Google Scholar 

  17. Prinz H (2009) Hill coefficients, dose–response curves and allosteric mechanisms. J Chem Biol 3:37–44. https://doi.org/10.1007/s12154-009-0029-3

    Article  PubMed  PubMed Central  Google Scholar 

  18. Ramsay RR, Tipton KF (2017) Assessment of enzyme inhibition: a review with examples from the development of monoamine oxidase and cholinesterase inhibitory drugs. Mol Basel Switz. https://doi.org/10.3390/molecules22071192

    Article  Google Scholar 

  19. Rasmussen DH, MacKenzie AP (1968) Phase diagram for the system water-dimethylsulphoxide. Nature 220:1315–1317

    CAS  Article  Google Scholar 

  20. Ruangchan N, Tongsook C, Sucharitakul J, Chaiyen P (2011) pH-dependent studies reveal an efficient hydroxylation mechanism of the oxygenase component of p-Hydroxyphenylacetate 3-Hydroxylase. J Biol Chem 286:223–233. https://doi.org/10.1074/jbc.M110.163881

    CAS  Article  PubMed  Google Scholar 

  21. Smirnovienė J, Smirnovas V, Matulis D (2017) Picomolar inhibitors of carbonic anhydrase: importance of inhibition and binding assays. Anal Biochem 522:61–72. https://doi.org/10.1016/j.ab.2017.01.022

    CAS  Article  PubMed  Google Scholar 

  22. Straus OH, Goldstein A, With the Technical Assistance of Plachte FL (1943). Zone behavior of enzymes. J Gen Physiol 26:559–585. https://doi.org/10.1085/jgp.26.6.559

  23. Strelow J, Dewe W, Iversen PW, Brooks HB, Radding JA, McGee J, Weidner J (2004). Mechanism of action assays for enzymes. In: Sittampalam GS, Grossman A, Brimacombe K, Arkin M, Auld D, Austin CP, Baell J, Bejcek B, Caaveiro JMM, Chung TDY, Coussens NP, Dahlin JL, Devanaryan V, Foley TL, Glicksman M, Hall MD, Haas JV, Hoare SRJ, Inglese J, Iversen Philip W, Kahl SD, Kales SC, Kirshner S, Lal-Nag M, Li Z, McGee J, McManus O, Riss T, Saradjian P, Trask OJ, Weidner JR, Wildey MJ, Xia M, Xu X (Eds.), Assay guidance manual. Eli Lilly & Company and the National Center for Advancing Translational Sciences, Bethesda, MD

  24. Todd MJ, Gomez J (2001) Enzyme kinetics determined using calorimetry: a general assay for enzyme activity? Anal Biochem 296:179–187. https://doi.org/10.1006/abio.2001.5218

    CAS  Article  PubMed  Google Scholar 

  25. Tonge PJ (2019) Quantifying the interactions between biomolecules: guidelines for assay design and data analysis. ACS Infect Dis 5:796–808. https://doi.org/10.1021/acsinfecdis.9b00012

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. Walsh R (2018) Comparing enzyme activity modifier equations through the development of global data fitting templates in Excel. PeerJ 6:e6082. https://doi.org/10.7717/peerj.6082

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. Wasserstein RL, Lazar NA (2016) The ASA statement on p -values: context, process, and purpose. Am Stat 70:129–133. https://doi.org/10.1080/00031305.2016.1154108

    Article  Google Scholar 

  28. Weiss JN (1997) The Hill equation revisited: uses and misuses. FASEB J 11:835–841. https://doi.org/10.1096/fasebj.11.11.9285481

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. Williams JW, Morrison JF (1979) [17] The kinetics of reversible tight-binding inhibition. In: Methods in enzymology, enzyme kinetics and mechanism part a initial rate and inhibitor methods. Academic Press, pp 437–467. https://doi.org/10.1016/0076-6879(79)63019-7

  30. Zhao P, Geyer RR, Boron WF (2017) A novel stopped-flow assay for quantitating carbonic-anhydrase activity and assessing red-blood-cell hemolysis. Front Physiol. https://doi.org/10.3389/fphys.2017.00169

    Article  PubMed  PubMed Central  Google Scholar 

Download references


Authors acknowledge the COST project ARBRE-MOBIEU CA15126.

Author information



Corresponding author

Correspondence to Daumantas Matulis.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation


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