A simple linearization method unveils hidden enzymatic assay interferences

• Tipo: Artigo em Revista Científica Internacional
• Data: 2019

Autores

Maria Filipa Pinto (Autor) FEUP; Jorge Ripoll-Rozada (Autor) Outra; Helena Ramos (Autor) Outra; Emma E. Watson (Autor) Outra; Charlotte Franck (Autor) Outra; Richard J. Payne (Autor) Outra; Lucília Saraiva (Autor) Outra; Pedro José Barbosa Pereira (Autor) Outra; Annalisa Pastore (Autor) Outra; Fernando Rocha (Autor) FEUP; Pedro M. Martins (Autor) Outra

Revista

Título: Biophysical Chemistry

Vol. 252 Nº Article number 106193

ISSN: 0301-4622

Editora: Elsevier

Indexação

ISI Web of Knowledge - 0 Citações

Scopus - 4 Citações

Projetos Associados

LEPABE - Projeto Estratégico do LEPABE - Laboratório de Engenharia de Processos, Ambiente, Biotecnologia e Energia

LEPABE - Laboratório de Engenharia de Processos, Ambiente, Biotecnologia e Energia

LEPABE-2-ECO-INNOVATION - LEPABE-2-ECO-INNOVATION

Outras Informações

ID Authenticus: P-00Q-PHJ

DOI: 10.1016/j.bpc.2019.106193

Abstract (EN): 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 a-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.

Idioma: Inglês

Tipo (Avaliação Docente): Científica

Nº de páginas: 15