Identification and isolation of two rat serum proteins with A-esterase activity toward paraoxon and chlorpyrifos-oxon

The active metabolites (oxons) of phosphorothionate insecticides can be detoxified via A-esterase hydrolysis. Two enzymes with A-esterase activity have been isolated from rat serum. Whole serum was applied to anion exchange gel (DEAF Sepharose Fast Flow) and incubated (1 hr). Tris-HCl buffer (0.05 M; pH 7.7, at 5 degrees) containing 0.25 M NaCl was added to the slurry and incubated. The decant, containing low A-esterase activity but a high protein concentration, was discarded. Further displacement of A-esterase from DEAF gel was achieved with 1.0 M NaCl in 0.05 M Tris-HCl buffer (pH 7.7 at 5 degrees). Following desalting and concentration, further separation was achieved by gel filtration (Sephacryl S-100 HR) and two sequential preparative scale isoelectric focusings. Final fractions contained two proteins of high molecular mass (one about 200 kDa and one between 137 and 200 kDa). The apparent range of isoelectric points for the two enzymes was 41.5 to 5.6. Following native-PAGE analysis, activity stains with beta-naphthyl acetate and Fast Garnet GBC in the presence of paraoxon (10(-5) M) verified that A esterase activity was associated with both proteins. Spectrophotometric assay detected A-esterase activity toward paraoxon, chlorpyrifos-oxon, and phenyl acetate in the final preparation.