Purification and characterization of paraoxon hydrolase from rat liver

Abstract

Paraoxonase (paraoxon hydrolase), an enzyme that hydrolyses paraoxon (O,O-diethyl O-p-nitrophenyl phosphate), is located in mammals primarily in the serum and liver. Although considerable information is available regarding serum paraoxonase, little is known about the hepatic form of this enzyme. The present work represents the first study on the purification of rat liver paraoxonase. This enzyme has been purified 415-fold to apparent homogeneity with a final specific activity of 1370 units/mg using a protocol consisting of five steps: solubilization of the microsomal fraction, hydroxyapatite adsorption, chromatography on DEAE-Sepharose CL-6B, non-specific affinity chromatography on Cibacron Blue 3GA and anion exchange on Mono Q HR 5/5. The presence of Ca2+ and Triton X-100 in the buffers throughout the purification procedure was essential for maintaining enzyme activity. SDS/PAGE of the final preparation indicated a single protein-staining band with an apparent Mr of 45 000. N-terminal and internal amino acid sequences were determined and compared with those of paraoxonases from human and rabbit serum and mouse liver, showing a high similarity. The pH profile showed optimum activity at pH 8.5. The pH stability and heat inactivation of the enzyme were also studied. The Km for liver paraoxonase was 1.69 mM.

Measurement of serum paraoxonase-1 activity in the evaluation of liver function

“…Essentially, the process consisted of the preparation of microsomes, solubilization with Triton X-100, adsorption on to hydroxyapatite, and chromatography with DEAE-52 cellulose to yield a 77-fold purified product. Later, Huang et al [23] isolated PON1 from mouse hepatic microsomes, and Rodrigo et al [24] in 1997, purified rat liver PON1 to homogeneity. They achieved a 415-fold purified product by using hydroxyapatite adsorption followed by three chromatography steps including DEAE-Sepharose, affinity chromatog raphy, and Mono Q HR fastperformance liquid chromatography.…”

Proteomic Analysis of Membrane-Associated Proteins from Rat Liver Autophagosomes

“…24; 9x enriched), 187 an arylesterase that detoxifies insecticide organophosphates and that also may protect agianst cardiovascular disease by metabolizing homocysteine thiolactone and by suppressing LDL oxidation. 188 Although paraoxonase is primarily a plasma protein secreted by the liver, it has also been found to be associated with liver microsomes, 189 where it could exert a protective function by hydrolyzing lipid peroxides generated during oxidative stress.…”

Purification of bovine serum paraoxonase and its immobilization on Eupergit C 250 L by covalent attachment

“…Further studies on this enzyme improved the purification procedures 25,26 . In our study, bovine serum PON1 was purified by using only two sequential steps: ammonium sulfate precipitation and HIC.…”