A pH sensitive colorometric assay for the high-throughput screening of enzyme inhibitors and substrates: A case study using kinases

Eli Chapman, Chi Huey Wong

Research output: Contribution to journalArticlepeer-review

44 Scopus citations

Abstract

We have developed an uncoupled, pH sensitive kinase assay that can be used for high-throughput screening of potential inhibitors or for determining substrate specificity. Kinases catalyze the transfer of a γ-phosphoryl group from ATP to an appropriate hydroxyl acceptor with the release of a proton. This assay is based on the detection of this proton using an appropriately matched buffer/indicator system. The assay was used to measure the activity of four readily available kinases, including hexokinase, glucokinase, glycerokinase, and pyruvate kinase, which was run in the reverse direction. We also went on to screen a small series of mono- and diphosphonucleotides for inhibition of hexokinase as well as a modest set of potential hexokinase substrates. We determined sucrose to be a modest substrate for hexokinase with a Km) of 1.8±0.2 mM, a kcat of 142±3 min-1, and a Vmax that is 15% of that for glucose. Given the importance of kinases in a diverse array of biological functions and disease states, there is a need for a simple, rapid assay system. We feel this assay will lend itself well to meet this end. This method should be applicable to many other enzymatic reactions which involve a change in pH.

Original languageEnglish (US)
Pages (from-to)551-555
Number of pages5
JournalBioorganic and Medicinal Chemistry
Volume10
Issue number3
DOIs
StatePublished - Feb 11 2002
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmaceutical Science
  • Drug Discovery
  • Clinical Biochemistry
  • Organic Chemistry

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