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.

A highly efficient magnetic solid acid nanocatalyst for the synthesis of new bulky heterocyclic compounds

“…11 This compound has been investigated for various applications in biomedical sciences including detoxication of biological uids, anti-cancer drug delivery, food analysis, environmental treatment and magnetic resonance imaging (MRI). [12][13][14][15][16][17][18][19] The magnetic iron oxide NPs have intrinsic instability over longer periods, because they have hydrophobic surfaces and in the absence of any proper surface coating, and the hydrophobic interactions between the NPs will cause them to aggregate and therefore, form large clusters, resulting in increased particle size and to reduce the surface energy. 20 Also magnetic particles lose their magnetism over time because bare iron oxide nanoparticles are easily oxidized in the air because of their high chemical activity, especially Fe 3 O 4 and g-Fe 2 O 3 NPs.…”

The effect of mercury, copper, and zinc on paraoxonase (PON) enzyme activity in Bonito (Sarda sarda) fish

“…When looked the effect of Hg +2 and Cu +2 heavy metal ions on PON enzyme activity studied in bonito (S. sarda) fish, it is seen that it causes a decrease in enzyme activity similar to the PON1 enzyme activity in rats. Samra et al (2010), examined the in vitro inhibitor effects of some metal ions, at 1.0 mM concentration on human PON1 enzyme activity. It was determined that Mg +2 and Mn +2 ions did not show any effect on human PON1 enzyme activity, Pb +2 , Co +2 , and Zn +2 ions decreased the activity, while Ni +2 , Cd +2, and Cu +2 ions inhibited the PON1 enzyme activity (Samra et al, 2010).…”

“…Samra et al (2010), examined the in vitro inhibitor effects of some metal ions, at 1.0 mM concentration on human PON1 enzyme activity. It was determined that Mg +2 and Mn +2 ions did not show any effect on human PON1 enzyme activity, Pb +2 , Co +2 , and Zn +2 ions decreased the activity, while Ni +2 , Cd +2, and Cu +2 ions inhibited the PON1 enzyme activity (Samra et al, 2010). Dedeoğlu et al (2014), in a study, determined changes occurring in PON1 enzyme activity purified from bull semen in the presence of Cu +2 , Mn +2 , Cd +2 , Zn +2 , Ni +2 , and Pb +2 heavy metal ions in different cuvette concentrations.…”

Catalytic Characteristics of New Antibacterials Based on Hexahistidine-Containing Organophosphorus Hydrolase

“…Interestingly, other researchers have previously shown inhibition of the paraoxon-hydrolyzing enzyme (human paraoxonase 1, PON1) by antibiotics, including ampicillin (IC 50 = 85.9 mM), gentamicin (IC 50 = 1.26 mM), and kanamycin (IC 50 = 1.24 mM) [29]. That is, having a similar activity pattern as His 6 -OPH and being suggested as another prospective quorum quenching enzyme [7], PON1 seems to be much more sensitive to inhibition by such common antibiotics.…”