Synthesis of Cholesterol-Conjugated Magnetic Nanoparticles for Purification of Human Paraoxonase 1

Abstract

Human serum paraoxonase 1 (PON1) is known as an antioxidant and is also involved in the detoxification of many compounds. In this study, a novel purification strategy was employed to purify the PON1 by using cholesterol-conjugated magnetic nanoparticles. Magnetic nanoparticles were synthesized and conjugated with cholesterol through diazotized p-aminohippuric acid. In Fourier transform infrared spectrum of cholesterol-p-aminohippuric acid-Fe(3)O(4) nanoparticles, the appearance of peaks at 3,358.3, 1,645 cm(-1), and at 2,334.9 cm(-1) confirmed the conjugation. The molecular weight of purified PON1 was nearly 45 kDa on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and isoelectric point was 5.3. The specific activity was 438 U mg(-1) protein, and the purification fold was 515 with 73% yield. The K (m) values were 1.3 and 0.74 mM with paraoxon and phenyl acetate, respectively. Western blot of 2D-PAGE confirmed the homogeneity and stability of the enzyme. Mg(+2), Mn(+2), glycerol, (NH(4))(2)SO(4), PEG 6000, Triton X-100, and phenylmethylsulfonyl fluoride did not show any effect on activity. Pb(+2), Co(+2), Zn(2+), ethanol, beta-mercaptoethanol, and acetone reduced the activity while Ni(2+), Cd(2+), Cu(2+), iodoacetic acid, SDS, dimethylformamide, DMSO inhibited the activity. In vitro enzyme activity was slightly reduced by acetyl salicylic and acetaminophen and reduced 50% with amino glycosides and ampicillin antibiotics at concentrations of 0.6 and 30 mg ml(-1), respectively. This is the first report for the synthesis of cholesterol-conjugated magnetic nanoparticles for simple purification of PON1 enzyme.