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A simple linearization method unveils hidden enzymatic assay interferences.

Maria Filipa Pinto1, Jorge Ripoll-Rozada2, Helena Ramos3

  • 1Instituto 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.

Biophysical Chemistry
|June 14, 2019
PubMed
Summary

This study introduces a linearization method (LM) to detect assay interferences in enzyme bioassays, ensuring accurate drug target screening. The method enhances the reliability of enzymology results by rigorously selecting valid kinetic data.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Drug Discovery

Background:

  • Enzymes are crucial drug targets in pharmaceuticals.
  • Bioassays for enzyme modulators can suffer from time-dependent inactivation and inhibition.
  • Ignoring later reaction phases doesn't eliminate these assay interferences.

Purpose of the Study:

  • To propose a linearization method (LM) for detecting spurious changes in enzymatic activity.
  • To enable rigorous selection of valid kinetic data in enzyme bioassays.
  • To enhance the reproducibility and accuracy of enzymology results.

Main Methods:

  • Developed a linearization method (LM) using modified coordinates to analyze enzyme progress curves.
  • Utilized procaspase-3, caspase-3, and α-thrombin as model enzymes.
  • Applied critical analysis of full progress curves.

Main Results:

  • The linearization method (LM) effectively detects assay interferences, even when not initially suspected.
  • The method is highly sensitive to signal readout distortions.
  • Demonstrated that simply ignoring post-initial-rate phases is insufficient to remove interferences.

Conclusions:

  • The proposed linearization method (LM) improves the reliability of enzyme bioassays.
  • Accurate identification of assay interferences leads to more trustworthy enzymology data.
  • Critical analysis of progress curves aids in distinguishing artifacts from true enzymatic inhibition mechanisms.