Evolved stereoselective hydrolases for broad-spectrum G-type nerve agent detoxification

A preferred strategy for preventing nerve agents intoxication is catalytic scavenging by enzymes that hydrolyze them before they reach their targets. Using directed evolution, we simultaneously enhanced the activity of a previously described serum paraoxonase 1 (PON1) variant for hydrolysis of the toxic S(P) isomers of the most threatening G-type nerve agents. The evolved variants show ≤340-fold increased rates and catalytic efficiencies of 0.2-5?× 10(7)?M(-1) min(-1). Our selection for prevention of acetylcholinesterase inhibition also resulted in the complete reversion of PON1's stereospecificity, from an enantiomeric ratio (E)?< 6.3?× 10(-4) in favor of the R(P) isomer of a cyclosarin analog in wild-type PON1, to E > 2,500 for the S(P) isomer in an evolved variant. Given their ability to hydrolyze G-agents, these evolved variants may serve as broad-range G-agent prophylactics.     

High-throughput screening of enzyme libraries: thiolactonases evolved by fluorescence-activated sorting of single cells in emulsion compartments

Single bacterial cells, each expressing a different library variant, were compartmentalized in aqueous droplets of water-in-oil (w/o) emulsions, thus maintaining a linkage between a plasmid-borne gene, the encoded enzyme variant, and the fluorescent product this enzyme may generate. Conversion into a double, water-in-oil-in-water (w/o/w) emulsion enabled the sorting of these compartments by FACS, as well as the isolation of living bacteria cells and their enzyme-coding genes. We demonstrate the directed evolution of new enzyme variants by screening >10(7) serum paraoxonase (PON1) mutants, to yield 100-fold improvements in thiolactonase activity. In vitro compartmentalization (IVC) of single cells, each carrying >10(4) enzyme molecules, in a volume of <10 femtoliter (fl), enabled detection and selection despite the fast, spontaneous hydrolysis of the substrate, the very low initial thiolactonase activity of PON1, and the use of difusable fluorescent products.     

Sensitive Assay for the Lactonase Activity of Serum Paraoxonase 1 (PON1) by Harnessing the Fluorescence Turn-On Characteristics of Bioorthogonally Synthesized and Geometrically Controlled Chemical Probes

The lactonase activity of paraoxonase 1 (PON1) has a crucial antiatherogenic function, and also serves as an important biochemical marker in human blood because the aberrant lactonase activity of PON1 is a key indicator for a number of diverse human diseases. However, no sensitive fluorescence assays that detect PON1 lactonase activity are available. We report the synthesis of two fluorescence turn-on chemical probes 16a and 16b (16) able to quantify PON1 lactonase activity. The chemical probes were constructed utilizing a disulfide-containing bicyclononyne, derivatives of rhodamine B and carboxyfluorescein, and reactions including copper-free azide–alkyne cycloaddition. Fluorescence quenching in 16 was characterized by spectroscopic studies and was mainly attributed to the effect of contact quenching. Kinetic analysis of 16b confirmed the outstanding reactivity and specificity of 16b with thiols in the presence of general base catalysts. The 16b-based assay was employed to determine PON1 lactonase activity, with a linear range of 10.8–232.1 U L⁻¹ and detection limit (LOD) of 10.8 U L⁻¹, to quantify serum PON1 activity in human sera, and to determine the K_i of 20.9 μM for the 2-hydroxyquinoline inhibition of PON1 lactonase. We are employing 16b to develop high-throughput assays for PON1 lactonase activity.     

In Vitro Inhibition Effect of Some Dihydroxy Coumarin Compounds on Purified Human Serum Paraoxonase 1 (PON1)

Human serum paraoxonase 1 (PON1; EC 3.1.8.1) is a high-density lipoprotein associated, calcium-dependent enzyme that hydrolyses aromatic esters, organophosphates and lactones and can protect the low-density lipoprotein against oxidation. In this study, in vitro inhibition effect of some dihydroxy coumarin compounds namely 6,7-dihydroxy-3-(2-methylphenyl)-2H-chromen-2-one (A), 6,7-dihydroxy-3-(3-methylphenyl)-2H-chromen-2-one (B) and 6,7-dihydroxy-3-(4-methylphenyl)-2H-chromen-2-one (C) on purified PON1 were investigated by using paraoxon as a substrate. PON1 was purified using two-step procedures, namely ammonium sulphate precipitation and Sepharose-4B-l-tyrosine-1-naphthylamine hydrophobic interaction chromatography. The purified enzyme had a specific activity of 11.76?U/mg. The dihydroxy coumarin derivatives of A and B compounds inhibited PON1 enzyme activity in a noncompetitive inhibition manner with K _i of 0.0080?±?0.256 and 0.0003?±?0.018?mM values, respectively. C compound exerted an uncompetitive inhibition of PON1 enzyme activity with K _i of 0.0010?±?0.173?mM. Moreover, dihydroxy coumarin derivatives of A, B and C compounds were effective inhibitors on purified human serum PON1 activity with IC₅₀ of 0.012, 0.022 and 0.003?mM values, respectively. IC₅₀ value of unsubstituted 6,7 dihydroxy coumarin was found as 0.178?mM. The present study has demonstrated that PON1 activity is very highly sensitive to studied coumarin derivatives.     

Stishovite's Relative: A Post‐Coesite Form of Phosphorus Oxonitride

Phosphorus oxonitride (PON) is isoelectronic with SiO₂ and may exhibit a similar broad spectrum of intriguing properties as silica. However, PON has only been sparsely investigated under high‐pressure conditions and there has been no evidence on a PON polymorph with a coordination number of P greater than 4. Herein, we report a post‐coesite (pc) PON polymorph exhibiting a stishovite‐related structure with P in a (5+1) coordination. The pc‐PON was synthesized using the multianvil technique and characterized by powder X‐ray diffraction, solid‐state NMR spectroscopy, TEM measurements and in situ synchrotron X‐ray diffraction in diamond anvil cells. The structure model was verified by single‐crystal X‐ray diffraction at 1.8 GPa and the isothermal bulk modulus of pc‐PON was determined to K₀=163(2) GPa. Moreover, an orthorhombic PON polymorph (o‐PON) was observed under high‐pressure conditions and corroborated as the stable modification at pressures above 17 GPa by DFT calculations.     

Evaluation of carbonic anhydrase and paraoxonase inhibition activities and molecular docking studies of highly water-soluble sulfonated phthalocyanines

The investigation of carbonic anhydrase and paraoxonase enzyme inhibition properties of water-soluble zinc and gallium phthalocyanine complexes ( 1 and 2 ) are reported for the first time. The binding of p-sulfonylphenoxy moieties to the phthalocyanine structure favors excellent solubilities in water, as well as providing an inhibition effect on carbonic anhydrase (CA) I and II isoenzymes and paraoxonase (PON1) enzyme. According to biological activity results, both complexes inhibited hCA I, hCA II, and PON1. Whereas 1 and 2 showed moderate hCA I and hCA II (off-target cytosolic isoforms) inhibitory activity (Ki values of 26.09 µM and 43.11 µM for hCA I and 30.95 µM and 33.19 µM for hCA II, respectively), they exhibited strong PON1 (associated with high-density lipoprotein [HDL]) inhibitory activity (Ki values of 0.37 µM and 0.27 µM, respectively). The inhibition kinetics were analyzed by Lineweaver–Burk double reciprocal plots. It revealed that 1 and 2 were noncompetitive inhibitors against PON1, hCA I, and hCA II. These complexes can be more advantageous than other synthetic CA and PON inhibitors due to their water solubility. Docking studies were carried out to examine the interactions between hCA I, hCA II, and PON1 inhibitors and metal complexes at a molecular level and to predict binding energies.     

The Structure and Function of Paraoxonase-1 and Its Comparison to Paraoxonase-2 and -3

Serum paraoxonase-1 (PON1) is the most studied member of the group of paraoxonases (PONs). This enzyme possesses three enzymatic activities: lactonase, arylesterase, and paraoxonase activity. PON1 and its isoforms play an important role in drug metabolism as well as in the prevention of cardiovascular and neurodegenerative diseases. Although all three members of the PON family have the same origin and very similar amino acid sequences, they have different functions and are found in different locations. PONs exhibit substrate promiscuity, and their true physiological substrates are still not known. However, possible substrates include homocysteine thiolactone, an analogue of natural quorum-sensing molecules, and the recently discovered derivatives of arachidonic acid—bioactive δ-lactones. Directed evolution, site-directed mutagenesis, and kinetic studies provide comprehensive insights into the active site and catalytic mechanism of PON1. However, there is still a whole world of mystery waiting to be discovered, which would elucidate the substrate promiscuity of a group of enzymes that are so similar in their evolution and sequence yet so distinct in their function.     

Electropolymerized Manganese Tetraaminophthalocyanine Thin Films onto Platinum Ultramicroelectrode for the Electrochemical Detection of Peroxynitrite in Solution

Peroxynitrite, ONOO^? also so‐called PON, is a very powerful oxidant and cytotoxic agent produced in biological systems by the recombination of nitric oxide and superoxide anion radical. Most of the techniques for assaying PON (chemiluminescence, fluorescence, UV‐visible spectroscopy, immunochemistry and EPR ) use indirect methods relying on measurements of secondary species. We report in this study the calibration of a chemically modified Pt ultramicroelectrode by electropolymerized manganese tetraaminophthalocyanine film (MnTAPc) for the determination of PON in aqueous solution. The obtained result allows showing for the first time a real‐time calibration curve of the amperometric determination of stable PON in aqueous solution. The sensitivity of the sensor is 14.6 nA mM^?1 and its detection limit is 5 μM.     

A High‐Pressure Polymorph of Phosphorus Oxonitride with the Coesite Structure

The chemical and physical properties of phosphorus oxonitride (PON) closely resemble those of silica, to which it is isosteric. A new high‐pressure phase of PON is reported herein. This polymorph, synthesized by using the multianvil technique, crystallizes in the coesite structure. This represents the first occurrence of this very dense network structure outside of SiO₂. Phase‐pure coesite PON (coe‐PON) can be synthesized in bulk at pressures above 15 GPa. This compound was thoroughly characterized by means of powder X‐ray diffraction, DFT calculations, and FTIR and MAS NMR spectroscopy, as well as temperature‐dependent diffraction. These results represent a major step towards the exploration of the phase diagram of PON at very high pressures and the possibly synthesis of a stishovite‐type PON containing hexacoordinate phosphorus.     

Paraoxonase 1 and Non-Alcoholic Fatty Liver Disease: A Meta-Analysis

Oxidative stress is involved in the pathophysiology of nonalcoholic fatty liver disease (NAFLD). However, reliable biomarkers of NAFLD in relation to oxidative stress are not available. While paraoxonase 1 (PON1) is an antioxidant biomarker, there appears to be mixed data on PON-1 in patients with NAFLD. The aim of this meta-analysis was to assess the current data on PON1 activity (i.e., paraoxonase and arylesterase) in patients with NAFLD. A PubMed, CENTRAL, and Embase search identified 12 eligible articles. In the meta-analysis, the paraoxonase activity was low in patients with NAFLD (mean difference (MD) −27.17 U/L; 95% confidence interval (CI) −37.31 to −17.03). No difference was noted in the arylesterase activity (MD 2.45 U/L; 95% CI −39.83 to 44.74). In a subgroup analysis, the paraoxonase activity was low in biopsy-proven nonalcoholic steatohepatitis (MD −92.11 U/L; 95% CI −115.11 to −69.11), while the activity in NAFLD as diagnosed by ultrasonography or laboratory data was similar (MD −2.91 U/L; 95% CI −11.63 to 5.80) to that of non-NAFLD. In summary, the PON1, especially paraoxonase, activity could be a useful biomarker of NAFLD. Further studies are warranted to ascertain the relevance of PON1 measurements in patients with NAFLD.     

Synergistic effects of novolac-based char former with a phosphorus/nitrogen-containing flame retardant in polyamide 6

The synergistic effect of phosphorus oxynitride（PON） with a novolac-based char former modified by salification （NA-metal salt） on the flame retardance of polyamide 6（PA6） was investigated.For this purpose,various flame-retardant PA6 systems were melt-compounded with PON,PON/NA,PON/NA-V₂O₅ and PON/NA-Fe₂O₃,and their flame retardance was evaluated by measuring the limiting oxygen index（LOI） values and UL-94 vertical burning ratings.The results showed that,compared with the PA6/PON/NA system,the combination of two char formers（NA-V₂O₅,NA-Fe₂O₃） with PON could obviously improve the char formation and flame retardance of PA6.The flame retardance and cone calorimetric analyses showed the stronger synergism as well as the better flame retardant performance of PON/NA-Fe₂O₃ flame retardant system. The effects of different char formers on the flame retardance and thermal stability of this system were also discussed.     

Association of Paraoxonase 1 (PON1) polymorphisms with osteoporotic fracture risk in postmenopausal Korean women

There is increasing evidence of a biochemical link between lipid oxidation and bone metabolism. Paraoxonase 1 (PON1) prevents the oxidation of low-density lipoprotein (LDL) and metabolizes biologically active phospholipids in oxidized LDLs. Here, we performed association analyses of genetic variation in PON1 to ascertain its contribution to osteoporotic fractures (OFs) and bone mineral density (BMD). We directly sequenced the PON1 gene in 24 Korean individuals and identified 26 sequence variants. A large population of Korean postmenopausal women (n=1,329) was then genotyped for eight selected PON1 polymorphisms. BMD at the lumbar spine and femoral neck was measured using dual-energy X-ray absorptiometry. Lateral thoracolumbar (T4-L4) radiographs were obtained for vertebral fracture assessment, and the occurrence of non-vertebral fractures (i.e., wrist, hip, forearm, humerus, rib, and pelvis) was examined using self-reported data. Multivariate analyses showed that none of the polymorphisms was associated with BMD at either site. However, +5989A>G and +26080T>C polymorphisms were significantly associated with non-vertebral and vertebral fractures, respectively, after adjustment for covariates. Specifically, the minor allele of +5989A>G exerted a highly protective effect against non-vertebral fractures (OR=0.59, P=0.036), whereas the minor allele of +26080T>C was associated with increased susceptibility to vertebral fractures (OR=1.73, P=0.020). When the risk for any OFs (i.e., vertebral or non-vertebral) was considered, the statistical significance of both polymorphisms persisted (P=0.002-0.010). These results suggest that PON1 polymorphisms could be one of useful genetic markers for OF risk in postmenopausal women.     

A Xanthene-Based Mono-Anionic PON Ligand: Exploiting a Bulky,Electronically Unsymmetrical Donor in Main Group Chemistry

The synthesis of a novel mono-anionic phosphino-amide ligand based on a xanthene backbone is reported, togetherr with the corresponding Ga^I complex, (PON)Ga (PON = 4-(di(2,4,6-trimethylphenyl)phosphino)-5-(2,6-diisopropylanilido)-2,7-di-tert-butyl-9,9-dimethylxanthene). The solid-state structure of (PON)Ga (obtained from X-ray crystallography) reveals very weak O⋅⋅⋅Ga and P⋅⋅⋅Ga interactions, consistent with a R₂NGa fragment which closely resembles those found in one-coordinate amidogallium systems. Strong N-to-Ga π donation from the amido substituent is reflected in a very short N−Ga distance (1.961(2) Å), while the P⋅⋅⋅Ga contact (3.076(1) Å) is well outside the sum of the respective covalent radii. While the donor properties of the PON ligand towards Ga^I are highly unsymmetrical, oxidation to Ga^III leads to much stronger coordination of the pendant phosphine as shown by P−Ga distances which are up to 20 % shorter. From a steric perspective, the PON ligand is shown to be significantly bulkier than related β-diketiminate systems, a finding consistent with reactions of (PON)Ga towards O-atom sources that proceed without oligomerization. Despite this, the enhanced P-donor properties brought about by oxidation at gallium are not sufficient to quench the reactivity of the highly polar Ga−O unit. Instead, intramolecular benzylic C−H activation is observed across the Ga−O bond of a transient gallanone intermediate.     

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

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.     

Genotyping single-nucleotide polymorphisms of human genes involved in organophosphate detoxification by high-resolution melting

Paraoxonase-1 (PON1) and butyrylcholinesterase (BCHE) are natural bioscavengers of organophosphate acetylcholinesterase inhibitors in the human body, which can determine individual sensitivity to organophosphate toxicity. Interindividual differences in activity of PON1 (catalytic bioscavenger) and substrate specificity are strongly associated with the substitution of two amino acids: Leu/Met (L/M) at position 55 (rs854560) and Gln/Arg (Q/R) at position 192 (rs662). In the case of BCHE (stoichiometric bioscavenger) substitution, Ala/Thr (A/T) at position 539 produces the so-called “K-variant” of the enzyme (rs1803274). Threonine allele is often co-inherited with an atypical BCHE allele (rs1799807). The atypical variant of BCHE displays a lower affinity for cholinesterase inhibitors. Genotyping rs662 and rs1803274 single-nucleotide polymorphisms (SNP) by high-resolution melting (HRM) is facilitated by the nucleotide substitution A>G (G>A), which resulted in a changed number of hydrogen bonds in the PCR product and, consequently, shifted T _m. In the case of rs854560, genotyping is complicated by the nucleotide substitution T>A, which has no significant effect on the T _m of the PCR product. An addition of a small quantity of LL homozygote DNA into the reaction mixture before PCR discriminates the three genotypes by the melt curves due to different amounts of heteroduplexes formed in the LM and MM samples. HRM analysis can be applied for genotyping human rs854560, rs662, and rs1803274 SNPs.

Modulating Glycoside Hydrolase Activity between Hydrolysis and Transfer Reactions Using an Evolutionary Approach

The proteins within the CAZy glycoside hydrolase family GH13 catalyze the hydrolysis of polysaccharides such as glycogen and starch. Many of these enzymes also perform transglycosylation in various degrees, ranging from secondary to predominant reactions. Identifying structural determinants associated with GH13 family reaction specificity is key to modifying and designing enzymes with increased specificity towards individual reactions for further applications in industrial, chemical, or biomedical fields. This work proposes a computational approach for decoding the determinant structural composition defining the reaction specificity. This method is based on the conservation of coevolving residues in spatial contacts associated with reaction specificity. To evaluate the algorithm, mutants of α-amylase (TmAmyA) and glucanotransferase (TmGTase) from Thermotoga maritima were constructed to modify the reaction specificity. The K98P/D99A/H222Q variant from TmAmyA doubled the transglycosydation/hydrolysis (T/H) ratio while the M279N variant from TmGTase increased the hydrolysis/transglycosidation ratio five-fold. Molecular dynamic simulations of the variants indicated changes in flexibility that can account for the modified T/H ratio. An essential contribution of the presented computational approach is its capacity to identify residues outside of the active center that affect the reaction specificity.     

New copper(II) complexes including pyridine-2,5-dicarboxylic acid: synthesis,spectroscopic, thermal properties,crystal structure and how these complexes interact with purified PON 1 enzyme

We report the synthesis of two square-pyramidal copper(II) complexes, [Cu(2,5-pydc)(2-aepy)(H₂O)]·H₂O, 1, and [Cu(2,5-pydc)(2-ampy)(H₂O)]·H₂O, 2 (2-aepy = 2-(aminoethyl)pyridine, 2-ampy = 2-(aminomethyl)pyridine, 2,5-pydc = pyridine-2,5-dicarboxylic acid or isocinchomeronic acid). The synthesized complexes have been characterized by X-ray diffraction, FT-IR, elemental, and thermal analysis techniques. The crystal structure of 1 was established by X-ray analysis. Powder X-ray diffraction analysis showed that the complexes are pure. The inhibition of human serum paraoxonase 1 (PON 1, EC 3.1.8.1) enzyme with these complexes were investigated. We used diethyl 4-nitrophenyl phosphate as a substrate to measure the paraoxonase activity of PON 1 enzyme spectrophotometrically. Complexes 1 and 2 decreased the in vitro PON 1 activity with different inhibition mechanisms. Complexes 1 and 2 inhibited paraoxonase activity of this enzyme as competitively and noncompetitively, respectively.     

Efficient formation of multicompartment hydrogels by stepwise self-assembly of thermoresponsive ABC triblock terpolymers

The gelation behavior of a poly(ethylene-alt-propylene)-b-poly(ethylene oxide)-b-poly(N-isopropylacrylamide) (PON) triblock terpolymer and a poly(N-isopropylacrylamide)-b-poly(ethylene oxide)-b-poly(N-isopropylacrylamide) (NON) triblock copolymer was studied by rheology over the concentration range 1-5 wt %. In comparison to the NON copolymer, gelation of the PON terpolymer was achieved at a much lower concentration, with a much sharper sol-gel transition. This is due to a stepwise gelation of PON terpolymers involving micellization at room temperature and gelation at elevated temperatures. The separation of micellization and gelation leads to the formation of a two-compartment network as observed by cryoTEM. The results highlight the intricate and tunable nanostructures and new properties accessible from ABC terpolymer hydrogels.     

Inhibition of paraoxonase 1 by coumarin‐substituted N‐heterocyclic carbene silver(I), ruthenium(II) and palladium(II) complexes

We synthesized three coumarin‐substituted benzimidazolium chlorides and their silver(I), ruthenium(II) and palladium(II) N‐heterocyclic carbene (NHC) complexes. All compounds were characterized using appropriate spectroscopic techniques and elemental analyses. Single‐crystal X‐ray structure of a Pd(II)–NHC complex ( 6b ) was also determined. The inhibitory properties of all compounds were tested on the activity of human paraoxonase 1 (PON1). All complexes exhibited weaker inhibitory properties than their corresponding benzimidazolium salts except for complex 6b which is the most active inhibitor with an IC₅₀ value of 3.01 μM among the compounds reported in this study. A kinetic evaluation showed that this complex inhibits PON1 activity in a non‐competitive manner. Molecular docking studies were also performed for 6b in order to obtain more insight into the binding mode.     

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

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.