Purification human PON1Q192 and PON1R192 isoenzymes by hydrophobic interaction chromatography and investigation of the inhibition by metals

In this study, a new purification strategy for human PON1 enzyme was developed using two-step procedures, namely ammonium sulfate precipitation and sepharose-4B-l-tyrosine-9-aminophenantrene hydrophobic interaction chromatography. SDS polyacrylamide gel electrophoresis of the enzyme indicates a single band with an apparent MW of 43 kDa. Overall purification rate of our method was found 901-fold for R isoenzyme and 453-fold for Q isoenzyme. The Vmax and KM of the purified enzyme were determined for Q isoenzyme 55 EU and 0.599 mM and for R isoenzyme 50 EU and 0.492 mM, respectively. The in vitro effects of some heavy metals (Hg, Cd, Cu, Mn and Ni) were investigated on the purified human serum PON1Q and R isoenzyme, using paraoxon as substrate. Metals were more effective inhibitors on purified human serum PON1R192 activity than PON1Q192 activity. The kinetics of interaction of metals with the purified human serum PON1R192 and PON1Q192 indicated a different inhibition pattern. Kinetic constants KM, Vmax, and inhibition type were determined.