Activity assay based on the immobilized enzyme kallikrein and mass spectrometry

Carvalho, Daniella Romano De and Laurentino, Bruna Barbosa and Rocha, Camila Loreta and Kool, Jeroen and Somsen, Govert and Amstalden van Hove, Erika and Cardoso, Carmen Lúcia (2022) Activity assay based on the immobilized enzyme kallikrein and mass spectrometry. Frontiers in Analytical Science, 2. ISSN 2673-9283

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Abstract

Deregulated activity and expression of human kallikreins (KLKs) may be involved in various pathologies, so these enzymes are an attractive biological target for identifying molecules that can modulate KLK activity. This identification involves applying fast and efficient screening methods. This work describes an off-line assay with mass spectrometry (MS) detection that uses KLK immobilized on Sepharose-NHS as a micro-column configuration (IMER-KLK-Sepharose-NHS). The mass spectrometry used has an ion trap analyzer and electrospray ionization (EIS). The HPLC-MS method for quantifying KLK activity was developed. The enzymatic assay conditions were optimized, and the IMER-KLK-Sepharose-NHS kinetic parameter (KMapp = 15.48 ± 3 μmol L−1) was evaluated. Finally, the method was validated by using leupeptin as a reference inhibitor (IC50 = 0.85 ± 0.10 μmol L−1). The developed method was able to identify the reference inhibitor and can be an alternative for screening KLK inhibitors.

Item Type: Article
Subjects: STM Library > Chemical Science
Depositing User: Managing Editor
Date Deposited: 11 Jan 2023 09:27
Last Modified: 02 May 2024 09:06
URI: http://open.journal4submit.com/id/eprint/327

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