Format

Send to

Choose Destination
Biophys Chem. 2019 Sep;252:106193. doi: 10.1016/j.bpc.2019.106193. Epub 2019 May 29.

A simple linearization method unveils hidden enzymatic assay interferences.

Author information

1
Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, Porto, Portugal; Laboratório de Engenharia de Processos, Ambiente, Biotecnologia e Energia (LEPABE), Faculdade de Engenharia da Universidade do Porto (FEUP), Porto, Portugal; IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal; Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.
2
IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal; Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.
3
Laboratório Associado para a Química Verde (LAQV), Rede de Química e Tecnologia (REQUIMTE), Faculdade de Farmácia da Universidade do Porto (FFUP), Porto, Portugal.
4
School of Chemistry, The University of Sydney, Sydney, New South Wales 2006, Australia.
5
Maurice Wohl Clinical Neuroscience Institute, King's College London, London, England, UK.
6
Laboratório de Engenharia de Processos, Ambiente, Biotecnologia e Energia (LEPABE), Faculdade de Engenharia da Universidade do Porto (FEUP), Porto, Portugal.
7
Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, Porto, Portugal; IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal; Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal. Electronic address: pmartins@ibmc.up.pt.

Abstract

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.

PMID:
31195341
DOI:
10.1016/j.bpc.2019.106193

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center