Spectrophotometric determination of paraoxonase within mouse carrier red blood cells

A method has been developed to continuously measure paraoxonase activity spectrophotometrically in carrier red blood cells (RBCs) containing paraoxonase. This enzyme has a broad substrate specificity that includes parathion, paraoxon, soman, sarin, di-isopropyl fluorophosphate and many other organophosphorus compounds. Paraoxon is hydrolysed by paraoxonase to the less toxic 4-nitrophenol and diethyl phosphate. Determination of enzymic activity was based on the liberation of 4-nitrophenol in the presence of mouse RBCs. Paraoxonase was encapsulated within murine RBCs by hypotonic dialysis with subsequent resealing and annealing. The enzyme within resealed RBCs actively hydrolyses paraoxon in biological fluids to its less toxic metabolites. Paraoxonase incorporated within RBCs, like other enzymes, was found to be quite stable once encapsulated into RBCs and this formed the basis for this spectrophotometric method. Increasing absorbance at 400 nm indicated paraoxon hydrolysis and was the basis employed to determine enzymic activity. The Km of the enzyme within erythrocytes was 0.04 mM. This method offers a convenient, rapid and continuous way to monitor paraoxonase activity inside the carrier cell.