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

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Chromogenics
Butyrylcholinesterase
Endotoxins
Assays
Hot Temperature
Kinetics
Chromogenic Compounds
Boiling liquids
Hydrolysis
Rate constants
Catalyst activity
Buffers
Peptides
Substrates

Keywords

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

ASJC Scopus subject areas

  • Toxicology

Cite this

@article{95caf419af244fd694862d726c8c0e6b,
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.",
keywords = "Amidase, Butyrylcholinesterase, Chromogenic substrate, Endotoxin, p-Nitroacetanilide",
author = "Andrew Brawner and Hinrichs, {Steven Heye} and Larson, {Marilynn A} and Oksana Lockridge",
year = "2016",
month = "4",
day = "5",
doi = "10.1016/j.cbi.2016.02.015",
language = "English (US)",
volume = "249",
pages = "19--22",
journal = "Chemico-Biological Interactions",
issn = "0009-2797",
publisher = "Elsevier Ireland Ltd",

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TY - JOUR

T1 - Heat pretreatment eliminates spurious butyrylcholinesterase enhancement of endotoxin levels in the kinetic chromogenic assay

AU - Brawner, Andrew

AU - Hinrichs, Steven Heye

AU - Larson, Marilynn A

AU - Lockridge, Oksana

PY - 2016/4/5

Y1 - 2016/4/5

N2 - 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.

AB - 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.

KW - Amidase

KW - Butyrylcholinesterase

KW - Chromogenic substrate

KW - Endotoxin

KW - p-Nitroacetanilide

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U2 - 10.1016/j.cbi.2016.02.015

DO - 10.1016/j.cbi.2016.02.015

M3 - Article

VL - 249

SP - 19

EP - 22

JO - Chemico-Biological Interactions

JF - Chemico-Biological Interactions

SN - 0009-2797

ER -