Purification of Holstein bull semen paraoxonase 1 (PON1) by hydrophobic interaction chromatography and investigation of its inhibition kinetics by heavy metals.

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.

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