Purification and Characterization of Paraoxonase 1 (PON1) from Swiss Black, Holstein, and Montofon Bovines

Abstract

Paraoxonase 1 (PON1: EC 3.1.8.1) is a calcium-dependent enzyme associated with high-density lipoproteins (HDLs) and has a protective effect against oxidation of low-density lipoproteins (LDLs) in mammals. PON1 is the best-studied member of a family of enzymes called serum paraoxonases, or PONs, identified in mammals and other vertebrates as well as in invertebrates. PONs exhibit a range of important activities, including drug metabolism and detoxification of organophosphates such as nerve agents. This study reports, for the first time, purification and biochemical characterization of serum PON1 from different bovine breeds namely Swiss Black, Holstein, and Montofon. Bovine serum PON1s were purified using ammonium sulfate precipitation followed by Sepharose-4B-L-tyrosine-1-naphthylamine hydrophobic interaction chromatography. SDS-polyacrylamide gel electrophoresis of the purified enzymes indicates a single band with an apparent MW of 43 kDa. The purified enzymes had a specific activity of 10.78, 27.00, and 22.38 U/mg for Swiss Black, Holstein, and Montofon bovines, respectively. The overall purification rates of our method were 262.47-, 2,476.90-, and 538.06-fold for Swiss Black, Holstein, and Montofon bovines, respectively. Furthermore, using phenyl acetate as a substrate, we determined the K M and V max values of the purified enzymes, as 0.80 mM, 1428.5 U/ml for Swiss Black; 0.40 mM, 714.3 U/ml for Holstein; and 0.50 mM, 1,111.1 U/ml for Montofon bovine. The present study has revealed that there is no substantial difference in PON1 activities among the studied bovine breeds.

A review of paraoxonase 1 properties and diagnostic applications

“…Less than about 5% of PON1 activity for both substrates was found in very low density lipoprotein (VLDLs) and low density lipoprotein (LDLs) fractions. In the Holstein Friesian, Japanese Blacks, Swiss Blacks and Montofon bovine breeds PON1 was identified as 43 kDa protein (Miyamoto et al 2005, Erzengin et al 2014.…”

Investigation of the possibilities of using PON1 enzyme activity in animal improvement

“…This value is higher compared to Holstein race dry (97.17) and the postpartum period (83.17) average PON1 activities and as well as the peak lactation period's (90.93) determined by Kulka et al [22]. On the other hand, this value is lower compared to Holstein-Friesian (178.69), Polish Red (168.54) and Norwegian (145.8) race average values determined by Erzengin et al [14] and Kulka et al [23]. On the other hand, the average PON1 enzyme activity determined in this study (144.42) was similar to the average Holstein race value (152.29) determined by Turk et al [24], while it was lower compared to pregnant Holstein cows in first 3 months of pregnancy (283), 3 to 6 months of pregnancy (257), intramammary infusion (216) pre-puerperal in nonpregnancy (223), late puerperal (189), and peak lactation (329) values determined in the same study [24].…”

“…PON enzyme also has a certain degree of antioxidant activity [10,11]. It has been suggested that on cattle, factors like race, age, productivity period, and health issues can cause changes on PON1 enzyme activity [12,13,14].…”

Induction by Phenobarbital of Phase I and II Xenobiotic-Metabolizing Enzymes in Bovine Liver: An Overall Catalytic and Immunochemical Characterization

“…Some of these studies represented the starting point for the evaluation of cattle DME activities. However, it should be underlined that some enzyme-substrate kinetic studies have already been published for bovines [ 97 , 104 , 105 , 106 , 107 , 108 ].…”