Identification of potential glycoprotein biomarkers in oral squamous cell carcinoma using sweet strategies.

The prevalence of oral squamous cell carcinoma (OSCC) is high in South and Southeast Asia regions. Most OSCC patients are detected at advanced stages low 5-year survival rates. Aberrant expression of glycosylated proteins was found to be associated with malignant transformation and cancer progression. Hence, identification of cancer-associated glycoproteins could be used as potential biomarkers that are beneficial for diagnosis or clinical management of patients. This study aims to identify the differentially expressed glycoproteins using lectin-based glycoproteomics approaches. Serum samples of 40 patients with OSCC, 10 patients with oral potentially malignant disorder (OPMD), and 10 healthy individuals as control group were subjected to two-dimensional gel electrophoresis (2-DE) coupled with lectin Concanavalin A and Jacalin that specifically bind to N- and O-glycosylated proteins, respectively. Five differentially expressed N- and O-glycoproteins with various potential glycosylation sites were identified, namely N-glycosylated α1-antitrypsin (AAT), α2-HS-glycoprotein (AHSG), apolipoprotein A-I (APOA1), and haptoglobin (HP); as well as O-glycosylated AHSG and clusterin (CLU). Among them, AAT and APOA1 were further validated using enzyme-linked immunosorbent assay (ELISA) (n = 120). It was found that AAT and APOA1 are significantly upregulated in OSCC and these glycoproteins are independent risk factors of OSCC. The clinical utility of AAT and APOA1 as potential biomarkers of OSCC is needed for further evaluation.

Interplay of isoform 1N4R tau protein and amyloid-ß peptide fragment 25-35 in reducing and non-reducing conditions.

Amyloid-ß (Aß) peptide and tau protein are two hallmark proteins in Alzheimer's disease (AD); however, the parameters, which mediate the abnormal aggregation of Aß and tau, have not been fully discovered. Here, we have provided an optimum method to purify tau protein isoform 1N4R by using nickel-nitrilotriacetic acid agarose chromatography under denaturing condition. The biochemical and biophysical properties of the purified protein were further characterized using in vitro tau filament assembly, tubulin polymerization assay, circular dichroism (CD) spectroscopy and atomic force microscopy. Afterwards, we investigated the effect of tau protein on aggregation of Aß (25-35) peptide using microscopic imaging and cell viability assay. Incubation of tau at physiologic and supra-physiologic concentrations with Aß25-35 for 40 days under reducing and non-reducing conditions revealed formation of two types of aggregates with distinct morphologies and dimensions. In non-reducing condition, the co-incubated sample showed granular aggregates, while in reducing condition, they formed annular protofibrils. Results from cell viability assay revealed the increased cell viability for the co-incubated sample. Therefore, the disassembling action shown by tau protein on Aß25-35 suggests the possibility that tau may have a protective role in preventing Aß peptide from acquiring the cytotoxic, aggregated form against oxidative stress damages.

Directed Evolution of a Cp*RhIII -Linked Biohybrid Catalyst Based on a Screening Platform with Affinity Purification.

Directed evolution of Cp*RhIII -linked nitrobindin (NB), a biohybrid catalyst, was performed based on an in vitro screening approach. A key aspect of this effort was the establishment of a high-throughput screening (HTS) platform that involves an affinity purification step employing a starch-agarose resin for a maltose binding protein (MBP) tag. The HTS platform enables efficient preparation of the purified MBP-tagged biohybrid catalysts in a 96-well format and eliminates background influence of the host E. coli cells. Three rounds of directed evolution and screening of more than 4000 clones yielded a Cp*RhIII -linked NB(T98H/L100K/K127E) variant with a 4.9-fold enhanced activity for the cycloaddition of acetophenone oximes with alkynes. It is confirmed that this HTS platform for directed evolution provides an efficient strategy for generating highly active biohybrid catalysts incorporating a synthetic metal cofactor.

Purification of protein therapeutics via high-affinity supramolecular host-guest interactions.

Efficient purification is crucial to providing large quantities of recombinant therapeutic proteins, such as monoclonal antibodies and cytokines. However, affinity techniques for manufacturing protein therapeutics that use biomolecule-conjugated agarose beads that harness specific biomolecular interactions suffer from issues related to protein denaturation, contamination and the need to maintain biomolecule-specific conditions for efficient protein capture. Here, we report a versatile and scalable method for the purification of recombinant protein therapeutics. The method exploits the high-affinity and controllable host-guest interactions between cucurbit[7]uril (CB[7]) and selected guests such as adamantylammonium. We show that the Herceptin (the brand name of trastuzumab, a monoclonal antibody drug used to treat breast cancer) and the much smaller cytokine interferon α-2a can be purified by site-specifically tagging them with adamantylammonium using the enzyme sortase A, followed by high-affinity binding with CB[7]-conjugated agarose beads and the recovery of the protein using a guest with a stronger affinity for CB[7]. The thermal and chemical stability of CB[7] beads and their scalability, recyclability and low cost may also make them advantageous for the manufacturing of biosimilars.

Recombinant Expression of Cyclotides Using Expressed Protein Ligation.

Cyclotides are naturally occurring microproteins (≈30 residues long) present in several families of plants. All cyclotides share a unique head-to-tail circular knotted topology containing three disulfide bridges forming a cystine knot topology. Cyclotides possess high stability to chemical, physical, and biological degradation and have been reported to cross cellular membranes. In addition, naturally occurring and engineered cyclotides have shown to possess various pharmacologically relevant activities. These unique features make the cyclotide scaffold an excellent tool for the design of novel peptide-based therapeutics by using molecular evolution and/or peptide epitope grafting techniques. In this chapter, we provide protocols to recombinantly produce a natively folded cyclotide making use of a standard bacterial expression system in combination with an intein-mediated backbone cyclization with concomitant oxidative folding.

Phospho-l-tyrosine-agarose chromatography: Adsorption of human IgG and its proteolytic fragments.

The behavior of human immunoglobulin G (IgG) and antigen-binding fragment (Fab fragment) adsorption onto phospho-l-tyrosine immobilized on agarose (P-Tyr-agarose) was evaluated by pseudoaffinity chromatography. The effects of buffer systems MES, MOPS, Bis-Tris, Tris-HCl and sodium phosphate (NaP) and pH on IgG adsorption were studied and high purity values were obtained (96%, based on ELISA analysis of albumin, transferrin and immunoglobulins A, G and M) when IgG was purified from human plasma diluted in 10 mmol L-1 NaP buffer at pH 6.0. The capture of IgG by the P-Tyr-agarose was also promising, since 91% of the IgG was adsorbed when plasma was diluted in 25 mmol L-1 MES buffer at pH 5.5, recommending its use for IgG depletion from human plasma under this condition. The experimental data on IgG adsorption kinetics were in agreement with the pseudo-second-order model. The adsorption isotherm data were well described by the Langmuir-Freundlich model with the value of parameter n being <1 (0.72), indicating negative cooperativity. Selectivity was achieved on P-Tyr-agarose from digested human IgG in HEPES 25 mmol L-1 buffer at pH 7.0 where Fab fragments were obtained in eluted fractions without Fc fragments (but with uncleaved IgG) with 86.2% recovery.

Selective Extraction and Antioxidant Properties of Thiol-Containing Peptides in Soy Glycinine Hydrolysates.

A highly selective procedure to extract thiol-containing peptides (TCPs) from complicated soy glycinin hydrolysates (SGHs) was described. This procedure included the reduction of disulfide bonds by 1,4-dithiothreitol (DTT) and enrichment of TCPs through Thiopropyl-Sephrose 6B covalent chromatography. TCPs were confirmed using a strategy based on mass shift after differential alkylation of sulfhydryl groups with iodoacetamide and N-ethylmaleimide by matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF-MS). The antioxidant activities of TCPs were evaluated using chemical assays. DTT reduction increased the concentration of sulfhydryl groups from 1.8 µmol/g to 113.8 µmol/g. The efficiency of the extraction was improved by optimizing the loading of sample, extraction and desorption time and the content of desorption reagent. Both of the adsorption and desorption process were found to fit a pseudo-second order model. MALDI-TOF-MS showed that 36 of the 45 extracted peptides were TCPs. The EC50 of TCPs for DPPH, hydroxyl radical, and superoxide anion radical was 0.1, 1.49 and 0.084 mg/mL, respectively. The reducing power of TCPs (0.2 mg/mL) was of 0.375. These results suggest that the combination of DTT reduction and Thiopropyl-Sepharose 6B covalent chromatograph was a successful pathway to extract TCPs from SGHs and the TCPs could be used as potential antioxidants.

Liquid Biopsy for Investigation of Cancer DNA in Esophageal Adenocarcinoma: Cell-Free Plasma DNA and Exosome-Associated DNA.

Liquid biopsy of cancers is an area of increasing interest in medical practice for the surveillance, management, and potential detection of malignant cells, using minimally invasive collection of body fluids. A liquid biopsy is particularly useful for metastatic cancers, which may be difficult to be sampled by core biopsy, due to difficulty of access or an occult location. Access to DNA shed from esophageal adenocarcinoma can enable the detection of mutations confirming the presence of malignant cells or the evolution of clonal lines with altered treatment response profiles. In this chapter, we detail a method for the isolation of cell-free DNA from blood plasma and DNA associated with exosomes in blood from patients with esophageal adenocarcinoma.

Fractionation of Soluble Proteins Using DEAE-Sepharose, SP-Sepharose, and Phenyl Sepharose Chromatographies for Proteomics.

In order to simplify a complex mixture of soluble proteins from tissues, a protocol to fractionate samples prior to two-dimensional (2D) gel electrophoresis has been developed. These methods involve the use of DEAE-Sepharose, SP-Sepharose, and phenyl Sepharose chromatographic columns and the fractionation of the protein mixtures based on differential anionic, cationic, and hydrophobic properties of the proteins, respectively. Fractionation of the soluble proteins with DEAE-Sepharose can result in an increase in the number of detectable 2D gel spots. These gel spots are amenable to protein identification by using in-gel trypsin digestions, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and peptide mass fingerprinting. The DEAE-Sepharose column fractionation acts to partition soluble proteins from cell extracts. Similarly, a SP-Sepharose column can fractionate soluble proteins and increase the number of detectable gel spots. Lastly, fractionation of cell extract with a phenyl Sepharose column can also result in an increase in the number of detectable 2D gel spots. This chapter describes an easy, inexpensive way to fractionate soluble proteins and a way to better profile proteomes.

Comparison of Different Methods of Purification and Concentration in Production of Influenza Vaccine.

The overwhelming majority of influenza vaccines are prepared with the use of chicken embryo allantoic fluid. The presence of ovalbumin (this protein constitutes >60% total protein in the allantoic fluid) in the vaccine can lead to severe allergy. Hence, effective reduction of ovalbumin content is of crucial importance for vaccine production. We compared two methods of purification and concentration of influenza virus: zonal gradient ultracentrifugation and combined ultrafiltration/diafiltration and exclusion chromatography protocol, used for fabrication of seasonal vaccines. Combined chromatography is comparable with zonal centrifugation protocol by the results of ovalbumin removal (to meet standard requirements).

Sequential Immunoprecipitation of Secretory Vesicle Proteins from Biosynthetically Labelled Cells.

Pulse radiolabelling of cells with radioactive amino acids is a common method for studying the biosynthesis of proteins. The labelled proteins can then be immunoprecipitated and analysed by electrophoresis and imaging techniques. This chapter presents a protocol for the biosynthetic labelling and immunoprecipitation of pancreatic islet proteins which are known to be affected in psychiatric disorders such as schizophrenia.

Native Folding of a Recombinant gpE1/gpE2 Heterodimer Vaccine Antigen from a Precursor Protein Fused with Fc IgG.

A recombinant strain HCV1 (hepatitis C virus [HCV] genotype 1a) gpE1/gpE2 (E1E2) vaccine candidate was previously shown by our group to protect chimpanzees and generate broad cross-neutralizing antibodies in animals and humans. In addition, recent independent studies have highlighted the importance of conserved neutralizing epitopes in HCV vaccine development that map to antigenic clusters in E2 or the E1E2 heterodimer. E1E2 can be purified using Galanthis nivalis lectin agarose (GNA), but this technique is suboptimal for global production. Our goal was to investigate a high-affinity and scalable method for isolating E1E2. We generated an Fc tag-derived (Fc-d) E1E2 that was selectively captured by protein G Sepharose, with the tag being removed subsequently using PreScission protease. Surprisingly, despite the presence of the large Fc tag, Fc-d E1E2 formed heterodimers similar to those formed by GNA-purified wild-type (WT) E1E2 and exhibited nearly identical binding profiles to HCV monoclonal antibodies that target conserved neutralizing epitopes in E2 (HC33.4, HC84.26, and AR3B) and the E1E2 heterodimer (AR4A and AR5A). Antisera from immunized mice showed that Fc-d E1E2 elicited anti-E2 antibody titers and neutralization of HCV pseudotype viruses similar to those with WT E1E2. Competition enzyme-linked immunosorbent assays (ELISAs) showed that antisera from immunized mice inhibited monoclonal antibody binding to neutralizing epitopes. Antisera from Fc-d E1E2-immunized mice exhibited stronger competition for AR3B and AR5A than the WT, whereas the levels of competition for HC84.26 and AR4A were similar. We anticipate that Fc-d E1E2 will provide a scalable purification and manufacturing process using protein A/G-based chromatography. IMPORTANCE: A prophylactic HCV vaccine is still needed to control this global disease despite the availability of direct-acting antivirals. Previously, we demonstrated that a recombinant envelope glycoprotein (E1E2) vaccine (genotype 1a) elicited cross-neutralizing antibodies from human volunteers. A challenge for isolating the E1E2 antigen is the reliance on GNA, which is unsuitable for large scale-up and global vaccine delivery. We have generated a novel Fc domain-tagged E1E2 antigen that forms functional heterodimers similar to those with native E1E2. Affinity purification and removal of the Fc tag from E1E2 resulted in an antigen with a nearly identical profile of cross-neutralizing epitopes. This antigen elicited anti-HCV antibodies that targeted conserved neutralizing epitopes of E1E2. Owing to the high selectivity and cost-effective binding capacity of affinity resins for capture of the Fc-tagged rE1E2, we anticipate that our method will provide a means for large-scale production of this HCV vaccine candidate.

Thermoresponsive Agarose Based Microparticles for Antibody Separation.

We report the development of thermoresponsive 4-mercaptoethylpyridine (MEP)-based chromatographic microsphere based resins for antibody separation that show switchable release abilities by adsorbing immunoglobulins at 40 °C and releasing the proteins at 5 °C. The thermoswitchable release properties were introduced to the porous resins by the grafting of linear poly(N-isopropylacrylamide) (PNIPAM) chains synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization, which were modified to possess MEP end functionalities. Adsorption of γ-globulins as a model antibody on the shortest PNIPAM-MEP (3 kDa) grafted microparticles display binding capacities of up to 20 g L(-1) at 40 °C and a significant decrease in binding capacity to less than 2.5 g L(-1) at 5 °C. By switching the temperature to 5 °C, the release of bound γ-globulins is shown to be as high as 90%. The effects of polymer chain length on the binding capacity are studied in detail and found to be critical as they influence the density of MEP functionalities on the particle surfaces.

Evolution and characterization of a benzylguanine-binding RNA aptamer.

Repurposing the "protein-labeling toolkit" for RNA research could be a pragmatic approach for developing new RNA-labeling methods. We have evolved an RNA aptamer that tightly binds benzylguanine (bG), the key ligand for the protein SNAP-tag. The aptamer tightly binds bG fluorophores and can be purified from cellular RNA with bG agarose under native conditions.

An alternative purification method for human serum paraoxonase 1 and its interactions with anabolic compounds.

In this study, an alternative purification method for human paraoxonase 1 (hPON1) enzyme was developed using two-step procedures, namely, ammonium sulfate precipitation and Sepharose-4B-L-tyrosine-3-aminophenantrene hydrophobic interaction chromatography. SDS-polyacrylamide gel electrophoresis of the enzyme indicates a single band with an apparent M(W) of 43 kDa. The enzyme was purified 219-fold with a final specific activity of 4,408,400 U/mg and a yield of 10%. Furthermore, we examined the in vitro effects of some anabolic compounds, such as zeranol, 17 ß-estradiol, diethylstilbestrol, oxytocin, and trenbolone on the enzyme activity to understand the better inhibitory properties of these molecules. The five anabolic compounds dose dependently decreased the activity of hPON1 with inhibition constants in the millimolar-micromolar range. The results show that these compounds exhibit inhibitory effects on hPON1 at low concentrations with IC50 values ranging from 0.064 to 16.900 µM.

Optimization extraction, preliminary characterization and antioxidant activity in vitro of polysaccharides from Stachys sieboldii Miq. tubers.

Response surface methodology was used to optimize the extraction conditions of water-soluble polysaccharides from Stachys sieboldii Miq. tubers. A central composite design was used to optimize the extraction processing parameters. The optimum extraction conditions are as follows: extraction temperature, 95°C; extraction time, 2.5h; water to raw material ratio, 16; and extraction frequency, 3. Under the optimized conditions, an experimental yield of 9.21 ± 0.18%, which is in good agreement with the predicted yield, was obtained. Purified polysaccharide SSP II-a was successfully obtained using diethylaminoethanol-Sepharose and Sepharose CL-6B column chromatography. SSP II-a was found to be an acidic polysaccharide fraction with an average molecular weight of 168kDa and composed of rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose. In vitro antioxidant activity assays suggested that SSP II-a presents high scavenging activity toward superoxide anion, hydroxyl, and 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) radicals but relatively lower scavenging activity toward 1,1-diphenyl-2-picrylhydrazyl radicals. The results indicated that response surface methodology is an effective method for the extraction of polysaccharides from S. sieboldii Miq. tubers and the polysaccharides could be explored as a potential antioxidant agent for use in medicine or functional food.

In vitro affinity reduction of biologic response modifiers from production buffy coat platelets exposed to recombinant protein receptors.

BACKGROUND: Recent studies link biologic response modifiers found in donor platelet (PLT) concentrates to transfusion reactions. We tested a novel method to deplete BRMs from PLT concentrates using apheresis. STUDY DESIGN AND METHODS: Whole blood from 25 donors was treated to yield PLTs for in vitro measurements on Days 2, 5, and 7. On Day 7, PLTs were filtrated through columns with either antibody-coated agarose or rh-megalin bound to antibody-coated agarose. In addition, we also tested the naked matrix (agarose) and another apheresis surface containing rh-cubilin bound to agarose. Megalin and cubilin are parts of the protein complex mediating BRM endocytosis in the human kidney. RESULTS: Compared to before filtration (951 × 10(9) ± 41 × 10(9) cells/L), PLT numbers decreased slightly after filtration over both naked (859 × 10(9) ± 38 × 10(9) ) and antibody-coated (848 × 10(9) ± 41 × 10(9) ) matrices (both p < 0.001 vs. before). Concentrations of interleukin (IL)-1ß, IL-12 (p40), IL-12 (p70), and IL-7 all decreased by approximately 40% even in the absence of a recombinant surface. After filtration over rh-cubilin, but not rh-megalin, concentrations of IFN-γ, IL-1ß, tumor necrosis factor-α, IL-12, and IL-7 all further decreased by 30% to 50%. CONCLUSION: In a pilot study of in vitro apheresis to deplete BRMs, we found that cell numbers and function remained largely unaffected by filtration. Significant reductions in BRMs occurred already with agarose. However, apheresis with the multiligand receptor rh-cubilin was able to further decrease concentrations.

Evaluación de la actividad antifúngica de extractos liquénicos e identificación de sus metabolitos / Evaluation of the fungicide activity from lichens extracts and identification their metabolites

Los líquenes son los hongos que establecen una relación simbiótica con un alga o cianobacteria. En esta simbiosis se producen por parte del hongo, una serie de metabolitos secundarios conocidos como sustancias liquénicas; las cuales presentan una marcada actividad antibiótica. En Cuba no se tienen antecedentes sobre estudios de metabolitos liquénicos por lo que se propone; evaluar el efecto fungicida de extractos liquénicos producidos por especies cubanas así como identificar sus metabolitos. Se emplearon líquenes de diferentes zonas del país (Parmotrema dilatatum, P. tinctorum, P. praesorediosum P. cristiferum, Ramalina americana, Cladonia ceratophylla y Cladonia portentosa spp. pacífica), a los cuales se les extrajo con acetona, las sustancias liquénicas almacenadas en el talo. Los extractos fueron probados contra los hongos fitopatógenos Rhizoctonia solani y Phythophtora nicotianae; por el método de envenenamiento del medio de cultivo agar papa dextrosa a concentraciones de: 0,01 por ciento; 0,03 por ciento y 0,07 por ciento. Se utilizó un control negativo de dimetilsufóxido al 0,07 por ciento y se determinaron los porcentajes de inhibición, cuyos resultados fueron analizados estadísticamente. Los metabolitos secundarios presentes en los extractos se identificaron por cromatografía de capa fina (TLC). Exceptuando el extracto liquénico de P. cristiferum, todos los demás mostraron más de un 50 por ciento de inhibición del crecimiento de ambos hongos a la concentración de 0,07 por ciento, mientras que a las restantes concentraciones los valores fueron variados con diferencias significativas con respecto al control. Se lograron identificar tres metabolitos liquénicos: metil 2‘-O- metilmicrofilinato, 4-O-Demetilmicrofilinico y el ácido ramaniloico.

Lichens are fungi that establish a symbiotic relationship with an alga or cyanobacterium. This symbiosis produced by the fungus, a series of secondary metabolites known as lichen substances; which show a strong antibiotic activity. In Cuba there is no background on the studies above lichen metabolites so it is proposed; evaluate the fungicidal activity of lichen extracts produced by Cuban species and to identify metabolites. Lichens from different areas of the country (Parmotrema dilatatum, P. tinctorum, P.praesorediosum, P. cristiferum, Ramalina americana, Cladonia ceratophylla and Cladonia portentosa spp. pacífica), to which it extracted with acetone, the lichen substances stored in tallus. The extracts were tested against the fungal pathogens Rhizoctonia solani and Phythophtora nicotianae; poisoning by the method of culture medium potato dextrose agar at concentrations of 0.01 percent; 0.03 percent and 0.07 percent. A negative control to 0.07 percent dimethylsulfoxide was used and the percentage of inhibition, the results were analyzed statistically determined. Secondary metabolites present in the extracts were identified by thin layer chromatography (TLC). Except P. cristiferum lichen extract, all others showed more than 50 percent growth inhibition of both fungi at concentration of 0.07 percent, while the remaining concentrations were varied values with significant differences from the control. Was made to identify three lichen metabolites metilmicrofilinato 2‘ methyl-O, 4-ODemetilmicrofilinico and ramaniloico acid.

Preparation, optimization and application of affinity absorbent with a polysaccharide YCP as the ligand.

YCP, an α-glucan from the mycelium of marine filamentous fungus Phoma herbarum YS4108, has great antitumor potential via enhancement of host immune through Toll-like receptor (TLR) 2 and TLR4 signaling. In the current study, YCP was coupled to EAH Sepharose 4B agarose beads to prepare the YCP-Sepharose affinity absorbent using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) as the activating agent. An orthogonal experiment L9 (3)(4) was applied to optimize the coupling procedure, giving the optimal parameters as follows: molar ratio of CDAP to YCP of 1:2, CDAP-activation time of 5 min, gel volume of 0.1 mL, and gel-incubation time of 72 h, respectively. Scanning electron microscopy analysis indicated successfully preparation of YCP immobilized sepharose beads, while these beads essentially maintained biological properties of free YCP since they can interact with TLR2 and TLR4 specifically at comparable level. Collectively, our findings provide an alternative approach to immobilize carbohydrate-based molecules for studying the carbohydrate-protein interaction.

Purification of Holstein bull semen paraoxonase 1 (PON1) by hydrophobic interaction chromatography and investigation of its inhibition kinetics by heavy metals.

In this study, paraoxonase 1 (PON1; EC 3.1.8.1) was purified from bull semen, and some characteristics of the enzyme were investigated. In vitro inhibition effect of some heavy metals, including Cu(2+), Mn(2+), Cd(2+), Zn(2+), Ni(2+), and Pb(2+), on the activity of the purified enzyme was also investigated. The purification of bull semen PON1 procedure was composed of two steps: ammonium sulfate precipitation and Sepharose-4B-L-tyrosine-1-naphthylamine hydrophobic interaction chromatography. The enzyme, having a specific activity of 288 EU/mg proteins, was purified 22.67-fold with a yield of 89 %. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the purified enzyme showed the presence of a single band with an apparent MW of 66 kDa. The V max and K M values for the paraoxon substrate were determined as 100 EU and 8.0 × 10(-5) M, respectively. The inhibitory effects of different heavy metals on PON1 activity were determined by using the paraoxon as a substrate. The results showed that all the metals, except for Cd(2+), inhibited the PON1 enzyme activity in a concentration-dependent fashion. IC50 values of Cu(2+), Mn(2+), Zn(2+), Ni(2+), and Pb(2+) were found as 2.59 × 10(-3), 1.17 × 10(-3), 42.74 × 10(-3), 99.10 × 10(-3), 48.80 × 10(-3) mM, respectively. Conversely, Cd(2+) increased the bull semen PON1 enzyme activity. The present study has demonstrated that Cu(2+), Mn(2+), Zn(2+), Ni(2+), and Pb(2+) are serious toxic metals, which are able to increase the risk of oxidative stress development and a subsequent decrease of semen quality.