title: Heat pretreatment eliminates spurious butyrylcholinesterase enhancement of endotoxin levels in the kinetic chromogenic assay.
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.
authors.affiliation: Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198, USA. Electronic address: andrew.brawner@unmc.edu.
authors.forename: Andrew
authors.initials: A
authors.lastname: Brawner
authors.affiliation: Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198, USA. Electronic address: shinrich@unmc.edu.
authors.forename: Steven H
authors.initials: SH
authors.lastname: Hinrichs
authors.affiliation: Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198, USA. Electronic address: malarson@unmc.edu.
authors.forename: Marilynn A
authors.initials: MA
authors.lastname: Larson
authors.affiliation: Eppley Institute, University of Nebraska Medical Center, Omaha, NE 68198, USA. Electronic address: olockrid@unmc.edu.
authors.forename: Oksana
authors.initials: O
authors.lastname: Lockridge
mesh_terms: D000814:Aniline Compounds / Q000378:metabolism; D001681:Biological Assay / Q000379:methods; D002091:Butyrylcholinesterase / Q000378:metabolism*; D004731:Endotoxins / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics
chemical_list: D000814:Aniline Compounds; D004731:Endotoxins; C019498:4-nitroaniline; D002091:Butyrylcholinesterase
keywords: Amidase; Butyrylcholinesterase; Chromogenic substrate; Endotoxin; p-Nitroacetanilide