2. Academic Validation
  3. Heat pretreatment eliminates spurious butyrylcholinesterase enhancement of endotoxin levels in the kinetic chromogenic assay

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

  • Chem Biol Interact. 2016 Apr 5:249:19-22. doi: 10.1016/j.cbi.2016.02.015.
Andrew Brawner 1,  Steven H Hinrichs 2,  Marilynn A Larson 3,  Oksana Lockridge 4
Affiliations

Affiliations

  • 1 Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198, USA. Electronic address: [email protected].
  • 2 Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198, USA. Electronic address: [email protected].
  • 3 Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198, USA. Electronic address: [email protected].
  • 4 Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198, USA. Electronic address: [email protected].
PMID: 26915974 DOI: 10.1016/j.cbi.2016.02.015
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.

Keywords

Amidase; Butyrylcholinesterase; Chromogenic substrate; Endotoxin; p-Nitroacetanilide.

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