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- Title
Purification of Holstein Bull Semen Paraoxonase 1 (PON1) by Hydrophobic Interaction Chromatography and Investigation of Its Inhibition Kinetics by Heavy Metals.
- Authors
Dedeoğlu, Nurcan; Arslan, Mikail; Erzengin, Mahmut
- 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, Mn, Cd, Zn, Ni, and Pb, 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 and K values for the paraoxon substrate were determined as 100 EU and 8.0 × 10 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, inhibited the PON1 enzyme activity in a concentration-dependent fashion. IC values of Cu, Mn, Zn, Ni, and Pb were found as 2.59 × 10, 1.17 × 10, 42.74 × 10, 99.10 × 10, 48.80 × 10 mM, respectively. Conversely, Cd increased the bull semen PON1 enzyme activity. The present study has demonstrated that Cu, Mn, Zn, Ni, and Pb are serious toxic metals, which are able to increase the risk of oxidative stress development and a subsequent decrease of semen quality.
- Subjects
METALS; HOLSTEIN-Friesian cattle; CHROMATOGRAPHIC analysis; AMINO acids; PROTEINS
- Publication
Biological Trace Element Research, 2014, Vol 158, Issue 1, p29
- ISSN
0163-4984
- Publication type
Article
- DOI
10.1007/s12011-014-9916-8