title: Purification of Holstein bull semen paraoxonase 1 (PON1) by hydrophobic interaction chromatography and investigation of its inhibition kinetics by heavy metals.
abstract: In this study, paraoxonase 1 (PON1; EC 3.1.8.1) was purified from bull semen, and some characteristics of the enzyme were investigated. In vitro inhibition effect of some heavy metals, including Cu(2+), Mn(2+), Cd(2+), Zn(2+), Ni(2+), and Pb(2+), on the activity of the purified enzyme was also investigated. The purification of bull semen PON1 procedure was composed of two steps: ammonium sulfate precipitation and Sepharose-4B-L-tyrosine-1-naphthylamine hydrophobic interaction chromatography. The enzyme, having a specific activity of 288 EU/mg proteins, was purified 22.67-fold with a yield of 89 %. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the purified enzyme showed the presence of a single band with an apparent MW of 66 kDa. The V max and K M values for the paraoxon substrate were determined as 100 EU and 8.0 × 10(-5) M, respectively. The inhibitory effects of different heavy metals on PON1 activity were determined by using the paraoxon as a substrate. The results showed that all the metals, except for Cd(2+), inhibited the PON1 enzyme activity in a concentration-dependent fashion. IC50 values of Cu(2+), Mn(2+), Zn(2+), Ni(2+), and Pb(2+) were found as 2.59 × 10(-3), 1.17 × 10(-3), 42.74 × 10(-3), 99.10 × 10(-3), 48.80 × 10(-3) mM, respectively. Conversely, Cd(2+) increased the bull semen PON1 enzyme activity. The present study has demonstrated that Cu(2+), Mn(2+), Zn(2+), Ni(2+), and Pb(2+) are serious toxic metals, which are able to increase the risk of oxidative stress development and a subsequent decrease of semen quality.
authors.affiliation: Faculty of Science and Letters, Department of Chemistry, Balikesir University, 10100, Balikesir, Turkey.
authors.forename: Nurcan
authors.initials: N
authors.lastname: Dedeoğlu
authors.forename: Mikail
authors.initials: M
authors.lastname: Arslan
authors.forename: Mahmut
authors.initials: M
authors.lastname: Erzengin
mesh_terms: D000645:Ammonium Sulfate / Q000737:chemistry; D000818:Animals; D043303:Aryldialkylphosphatase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D011232:Chemical Precipitation; D002847:Chromatography, Agarose / Q000379:methods*; D003300:Copper / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D007854:Lead / Q000494:pharmacology; D008297:Male; D008345:Manganese / Q000494:pharmacology; D019216:Metals, Heavy / Q000494:pharmacology*; D008970:Molecular Weight; D009532:Nickel / Q000494:pharmacology; D010261:Paraoxon / Q000378:metabolism; D012661:Semen / Q000201:enzymology*; D012685:Sepharose / Q000737:chemistry; D013379:Substrate Specificity; D015032:Zinc / Q000494:pharmacology
chemical_list: D019216:Metals, Heavy; D007854:Lead; D008345:Manganese; D003300:Copper; D009532:Nickel; D012685:Sepharose; D043303:Aryldialkylphosphatase; D015032:Zinc; D010261:Paraoxon; D000645:Ammonium Sulfate