Fluorescence anisotropy assay for the traceless kinetic analysis of protein digestion

Felix Cleemann, Peter Karuso

Research output: Contribution to journalArticleResearchpeer-review

Abstract

A novel fluorescence polarization assay based on the natural fluorophore epicocconone has been developed. This assay allows the rapid and accurate determination of enzyme kinetic parameters as well as inhibition constants through the measurement of fluorescence anisotropy on the actual substrate of the protease. It takes advantage of epicocconone's ability to reversibly react with proteins to form an internal charge-transfer complex that is highly fluorescent. The protein-substrate is labeled in situ without the need for prior incubation and/or derivatization steps, which saves time and effort compared to methods employing specifically labeled protein-substrates. The assay can be carried out in 96- or 384-well plates, making it suitable for high-throughput applications in drug development and biotechnology.

LanguageEnglish
Pages4170-4174
Number of pages5
JournalAnalytical Chemistry
Volume80
Issue number11
DOIs
Publication statusPublished - 1 Jun 2008

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Assays
Anisotropy
Fluorescence
Substrates
Enzyme inhibition
Enzyme kinetics
Proteins
Fluorophores
Biotechnology
Kinetic parameters
Charge transfer
Peptide Hydrolases
Throughput
Polarization
Pharmaceutical Preparations
epicocconone

Cite this

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Fluorescence anisotropy assay for the traceless kinetic analysis of protein digestion. / Cleemann, Felix; Karuso, Peter.

In: Analytical Chemistry, Vol. 80, No. 11, 01.06.2008, p. 4170-4174.

Research output: Contribution to journalArticleResearchpeer-review

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