SPECTROPHOTOMETRIC DETERMINATION OF PARAOXONASE WITHIN MOUSE CARRIER RED-BLOOD-CELLS

A method has been developed to continuously measure paraoxonase activi ty spectrophotometrically in carrier red blood cells (RBCs) containing paraoxonase. This enzyme has a broad substrate specificity that inclu des parathion, paraoxon, soman, sarin, di-isopropyl fluorophosphate an d many other organophosphorus compounds. Paraoxon is hydrolysed by par aoxonase to the less toxic 4-nitrophenol and diethyl phosphate. Determ ination of enzymic activity was based on the liberation of 4-nitrophen ol in the presence of mouse RBCs. Paraoxonase was encapsulated within murine RBCs by hypotonic dialysis with subsequent resealing and anneal ing. The enzyme within resealed RBCs actively hydrolyses paraoxon in b iological fluids to its less toxic metabolites. Paraoxonase incorporat ed within RBCs, like other enzymes, was found to be quite stable once encapsulated into RBCs and this formed the basis for this spectrophoto metric method. Increasing absorbance at 400 nm indicated paraoxon hydr olysis and was the basis employed to determine enzymic activity. The K (m) 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.