title: Purification human PON1Q192 and PON1R192 isoenzymes by hydrophobic interaction chromatography and investigation of the inhibition by metals.
abstract: 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 43kDa. Overall purification rate of our method was found 901-fold for R isoenzyme and 453-fold for Q isoenzyme. The V(max) and K(M) 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 PON1(R192) activity than PON1(Q192) activity. The kinetics of interaction of metals with the purified human serum PON1(R192) and PON1(Q192) indicated a different inhibition pattern. Kinetic constants K(M), V(max), and inhibition type were determined.
authors.affiliation: Balikesir University, Science and Art Faculty, Department of Chemistry/Biochemistry Section, 10100 Balikesir, Turkey. ngencer@balikesir.edu.tr
authors.forename: Nahit
authors.initials: N
authors.lastname: Gençer
authors.forename: Oktay
authors.initials: O
authors.lastname: Arslan
mesh_terms: D043303:Aryldialkylphosphatase / Q000037:antagonists & inhibitors* / Q000097:blood*; D002847:Chromatography, Agarose; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D020128:Inhibitory Concentration 50; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000097:blood*; D007700:Kinetics; D019216:Metals, Heavy / Q000494:pharmacology*; D010261:Paraoxon / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D007527:Isoenzymes; D019216:Metals, Heavy; D043303:Aryldialkylphosphatase; C468406:PON1 protein, human; D010261:Paraoxon