title: Separating esterase targets of organophosphorus compounds in the brain by preparative chromatography.
abstract: Low level exposure to organophosphorus esters (OPs) may cause long-term neurological effects and affect specific cognition domains in experimental animals and humans. Action on known targets cannot explain most of these effects by. Soluble carboxylesterases (EC 3.1.1.1) of chicken brain have been kinetically discriminated using paraoxon, mipafox and phenylmethyl sulfonylfluoride as inhibitors and phenyl valerate as a substrate. Three different enzymatic components were discriminated and called Eα, Eβ and Eγ. In this work, a fractionation procedure with various steps was developed using protein native separation methods by preparative HPLC. Gel permeation chromatography followed by ion exchange chromatography allowed enriched fractions with different kinetic behaviors. The soluble chicken brain fraction was fractionated, while total esterase activity, proteins and enzymatic components Eα, Eβ and Eγ were monitored in each subfraction. After the analysis, 13 fractions were pooled and conserved. Preincubation of the soluble chicken brain fraction of with the organophosphorus mipafox gave rise to a major change in the ion exchange chromatography profile, but not in the molecular exchanged chromatography profile, which suggest that mipafox permanently modifies the ionic properties of numerous proteins.
authors.affiliation: University "Miguel Hernandez" of Elche, Institute of Bioengineering, Unit of Toxicology and Chemical Safety, Alicante, Spain. Electronic address: imangas@umh.es.
authors.forename: I
authors.initials: I
authors.lastname: Mangas
authors.affiliation: University "Miguel Hernandez" of Elche, Institute of Bioengineering, Unit of Toxicology and Chemical Safety, Alicante, Spain.
authors.forename: E
authors.initials: E
authors.lastname: Vilanova
authors.affiliation: University "Miguel Hernandez" of Elche, Institute of Bioengineering, Unit of Toxicology and Chemical Safety, Alicante, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Benabent
authors.affiliation: University "Miguel Hernandez" of Elche, Institute of Bioengineering, Unit of Toxicology and Chemical Safety, Alicante, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Estévez
mesh_terms: D000818:Animals; D001921:Brain / Q000187:drug effects* / Q000201:enzymology*; D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification*; D005591:Chemical Fractionation; D002645:Chickens; D002800:Cholinesterase Inhibitors / Q000494:pharmacology*; D002845:Chromatography / Q000379:methods*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D007527:Isoenzymes; D007531:Isoflurophate / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D002800:Cholinesterase Inhibitors; D007527:Isoenzymes; D007531:Isoflurophate; C005238:mipafox; D002265:Carboxylic Ester Hydrolases
keywords: Carboxylesterases; Chromatography; Neurotoxicity targets; Organophosphorus compounds; Protein separation