Paraoxonase activity against nerve gases measured by capillary electrophoresis and characterization of human serum paraoxonase (PON1) polymorphism in the coding region (Q192R)

Abstract

An analytical method for determining paraoxonase activity against sarin, soman and VX was established. We used capillary electrophoresis to measure directly the hydrolysis products: alkyl methylphosphonates. After enzymatic reaction of human serum paraoxonase (PON1) with nerve gas, substrate was removed with dichloromethane, and alkyl methylphoshphonates were quantified by capillary electrophoresis of reversed osmotic flow using cationic detergent and sorbic acid. This method was applied to the characterization of human serum PON1 polymorphism for nerve gas hydrolytic activity in the coding region (Q192R). PON1-192 and PON1-55 genotypes were determined by their gel electrophoretic fragmentation pattern with restriction enzymes after polymerase chain reaction (PCR) of blood leukocyte genomic DNA. Frequencies of genotypes among 63 members of our institutes with PON1-192 and PON1-55 were 9.5% ( 192QQ), 30.1% ( 192QR) and 44.4% ( 192RR), and 82.5% ( 55LL), 17.5% ( 55LM) and 0% ( 55MM), respectively. 192Q and 192R enzymes were purified from the respective genotype human plasma, using blue agarose affinity chromatography and diethyl amino ethane (DEAE) anion exchange chromatography. V max and K m were measured using Lineweaver–Burk plots for hydrolytic activities against sarin, soman and VX at pH 7.4 and 25 °C. For sarin and soman, the V max for 192Q PON1 were 3.5- and 1.5-fold higher than those for 192R PON1; and k cat/K m for 192Q PON1 were 1.3- and 2.8-fold higher than those for 192R PON1. For VX, there was little difference in V max and k cat/K m between 192Q and 192R PON1, and VX hydrolyzing activity was significantly lower than those for sarin and soman. PON1 hydrolyzed sarin and soman more effectively than paraoxon.