2. Academic Validation
  3. Dephospho-CoA kinase provides a rapid and sensitive radiochemical assay for coenzyme A and its thioesters

Dephospho-CoA kinase provides a rapid and sensitive radiochemical assay for coenzyme A and its thioesters

  • Anal Biochem. 2007 Sep 1;368(1):17-23. doi: 10.1016/j.ab.2007.05.031.
Caryn Wadler 1,  John E Cronan
Affiliations

Affiliation

  • 1 Department of Microbiology, University of Illinois, Urbana, IL 61801, USA.
PMID: 17603993 DOI: 10.1016/j.ab.2007.05.031
Abstract

A new approach to determine in vivo pools of coenzyme A (CoA) and short chain acyl-CoA thioesters is reported. The metabolites released by extraction with trichloroacetic acid are recovered and quantitatively dephosphorylated by treatment with shrimp Alkaline Phosphatase. Following Phosphatase removal, the dephosphorylated CoA metabolites are quantitatively rephosphorylated by treatment with [gamma-33P]ATP plus a dephospho-CoA kinase. The resulting radioactive CoA metabolites are then separated by reverse-phase high-performance liquid chromatography and quantitated by scintillation counting. Due to the enzymatic radiophosphorylation, the assay is specific for CoA and its short chain thioesters and is sensitive to sub-picomole levels of these compounds.

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