Heat pretreatment eliminates spurious butyrylcholinesterase enhancement of endotoxin levels in the kinetic chromogenic assay

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Abstract

The kinetic chromogenic endotoxin assay measures the release of p-nitroaniline from the chromogenic peptide substrate Ac-IEAR-pNA. As part of our project to purify large quantities of human butyrylcholinesterase (HuBChE), we evaluated pure HuBChE for endotoxin levels. We found that HuBChE contributed up to 90% of the yellow p-nitroaniline product in a standard endotoxin assay through the catalytic hydrolysis of Ac-IEAR-pNA with a rate constant of 0.016 min-1 and a Km of 2.9 mM in potassium phosphate buffer pH 7.0 at 24 °C. Thus, endotoxin concentrations for native BChE are artificially high in the kinetic chromogenic assay. Destruction of HuBChE catalytic activity by boiling yields endotoxin concentrations that more accurately reflect the endotoxin concentration in purified HuBChE preparations.

Original languageEnglish (US)
Pages (from-to)19-22
Number of pages4
JournalChemico-Biological Interactions
Volume249
DOIs
StatePublished - Apr 5 2016

Keywords

  • Amidase
  • Butyrylcholinesterase
  • Chromogenic substrate
  • Endotoxin
  • p-Nitroacetanilide

ASJC Scopus subject areas

  • Toxicology

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