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Separating esterase targets of organophosphorus compounds in the brain by preparative chromatography

I Mangas1, E Vilanova1, M Benabent1

  • 1University "Miguel Hernandez" of Elche, Institute of Bioengineering, Unit of Toxicology and Chemical Safety, Alicante, Spain.

Toxicology Letters
|December 21, 2013
PubMed

Insights

Low-level organophosphorus ester (OP) exposure may cause neurological effects not explained by known targets. Chicken brain carboxylesterases were fractionated, revealing distinct enzymatic components potentially involved in these effects.

Area of Science:

  • Biochemistry
  • Neurotoxicology
  • Enzymology

Background:

  • Low-level organophosphorus ester (OP) exposure is linked to long-term neurological effects and cognitive deficits in humans and animals.
  • The precise mechanisms underlying these neurotoxic effects remain unclear, as they are not fully explained by interactions with known OP targets.

Purpose of the Study:

  • To kinetically discriminate and isolate soluble carboxylesterases (EC 3.1.1.1) from chicken brain.
  • To develop a fractionation procedure to separate these enzymatic components.
  • To investigate the impact of organophosphorus compounds on the properties of these enzymes.

Main Methods:

  • Kinetic discrimination of chicken brain soluble carboxylesterases using specific inhibitors (paraoxon, mipafox, phenylmethyl sulfonylfluoride) and a substrate (phenyl valerate).
  • Development of a multi-step fractionation procedure involving preparative HPLC, gel permeation chromatography, and ion exchange chromatography.
  • Monitoring of total esterase activity, protein content, and individual enzymatic components (Eα, Eβ, Eγ) throughout the fractionation process.

Main Results:

  • Three distinct enzymatic components (Eα, Eβ, Eγ) were kinetically discriminated in the soluble chicken brain fraction.
  • A fractionation procedure successfully enriched these components, yielding 13 pooled fractions with different kinetic behaviors.
  • Preincubation with the organophosphorus compound mipafox significantly altered the ion exchange chromatography profile, indicating a modification of protein ionic properties, while the molecular exchange chromatography profile remained unchanged.

Conclusions:

  • Chicken brain contains multiple soluble carboxylesterase components with distinct kinetic properties.
  • The developed fractionation method effectively separates these components.
  • Organophosphorus compounds like mipafox can induce significant changes in the ionic characteristics of brain proteins, suggesting a potential mechanism for their neurotoxic effects beyond direct target inhibition.
Keywords:
CarboxylesterasesChromatographyNeurotoxicity targetsOrganophosphorus compoundsProtein separation

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