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-Fe₃O₄ 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, (NH₄)₂SO₄, PEG 6000, Triton X-100, and phenylmethylsulfonyl fluoride did not show any effect on activity. Pb⁺², Co⁺², Zn²⁺, ethanol, β-mercaptoethanol, and acetone reduced the activity while Ni²⁺, Cd²⁺, Cu²⁺, 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.