A simple linearization method unveils hidden enzymatic assay interferences

Maria Filipa Pinto, Jorge Ripoll-Rozada, Helena Ramos, Emma E. Watson, Charlotte Franck, Richard J. Payne, Lucília Saraiva, Pedro José Barbosa Pereira, Annalisa Pastore, Fernando Rocha, Pedro M. Martins*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

6 Citations (Scopus)


Enzymes are among the most important drug targets in the pharmaceutical industry. The bioassays used to screen enzyme modulators can be affected by unaccounted interferences such as time-dependent inactivation and inhibition effects. Using procaspase-3, caspase-3, and α-thrombin as model enzymes, we show that some of these effects are not eliminated by merely ignoring the reaction phases that follow initial-rate measurements. We thus propose a linearization method (LM) for detecting spurious changes of enzymatic activity based on the representation of progress curves in modified coordinates. This method is highly sensitive to signal readout distortions, thereby allowing rigorous selection of valid kinetic data. The method allows the detection of assay interferences even when their occurrence is not suspected a priori. By knowing the assets and liabilities of the bioassay, enzymology results can be reported with enhanced reproducibility and accuracy. Critical analysis of full progress curves is expected to help discriminating experimental artifacts from true mechanisms of enzymatic inhibition.

Original languageEnglish
Article number106193
JournalBiophysical Chemistry
Early online date29 May 2019
Publication statusPublished - Sept 2019


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