In vitro efficacy of paraoxonase 1 from multiple sources against various organophosphates

Toxicology in Vitro 25 (2011) 905–913

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In vitro efficacy of paraoxonase 1 from multiple sources against various organophosphates Manojkumar Valiyaveettil a,⇑, Yonas Alamneh a, Lionel Biggemann a, Iswarduth Soojhawon a, Heba A. Farag a,b, Prashasthi Agrawal a,b, Bhupendra P. Doctor a, Madhusoodana P. Nambiar a,b,⇑ a b

Closed Head Injury Branch, Center for Military Psychiatry and Neuroscience, Walter Reed Army Institute of Research, Silver Spring, MD 20910, United States Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, United States

a r t i c l e

i n f o

Article history: Received 10 February 2011 Accepted 25 February 2011 Available online 5 March 2011 Keywords: Catalytic bioscavenger Paraoxonase 1 Organophosphates Protein purification Nerve agents Chemical defense

a b s t r a c t Paraoxonase 1 (PON1) has been described as a potential catalytic bioscavenger due to its ability to hydrolyze organophosphate (OP) insecticides and nerve agents. In vitro catalytic efficiency of purified human and rabbit serum PON1 against different OP substrates was compared to human recombinant PON1, expressed in Trichoplusia ni larvae. Highly purified human and rabbit serum PON1s were prepared by multiple chromatography methods. Purified enzymes showed higher catalytic activity with the substrate p-nitrophenyl acetate compared to diethyl paraoxon. The hydrolyzing potential of PON1s against multiple OPs was evaluated by using an in vitro acetylcholinesterase back-titration assay. Significant differences in the catalytic efficiency of all the three PON1s with regard to various OP substrates were observed. Purified PON1s showed higher catalytic activity towards diisopropylfluorophosphate followed by diethylparaoxon compared to dimethyl paraoxon. Heat inactivation or incubation of PON1 with specific inhibitor resulted in complete loss of the enzyme catalytic activity indicating that OP hydrolysis was intrinsic to PON1. In conclusion, purified PON1s from multiple sources show significant differences in the catalytic activity against several OP substrates. These results underscore the importance of systematic analysis of candidate PON1 molecules for developing as an effective catalytic bioscavenger against toxic OPs and chemical warfare nerve agents. Published by Elsevier Ltd.

1. Introduction The primary mode of action of various organophosphates (OPs) and chemical warfare nerve agents (CWNAs) is irreversible inhibition of the enzyme acetylcholinesterase (AChE) both in central and peripheral nervous systems (Aldridge and Davison, 1953; Shih et al., 2005). Accumulation of acetylcholine (ACh) leads to hyperch-

Abbreviations: PON1, paraoxonase 1; OPs, organophosphates; CWNAs, chemical warfare nerve agents; DFP, diisopropylfluorophosphate; DEP, diethyl paraoxon; DMP, dimethyl paraoxon; HPON1, human PON1; RPON1, rabbit PON1; rePON1, recombinant PON1; Apo AI, apolipoprotein AI; AChE, acetylcholinesterase; ACh, acetylcholine; HuBChE, human butyrylcholinesterase; Con A, concanavalin A; BCA, bicinchoninic acid; SDS–PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; PVDF, polyvinylidenedifluoride; ECL, enhanced chemiluminescence; PBST, phosphate buffered saline/0.1% Tween-20; p-NPA, p-nitrophenyl acetate; ATCh, acetylthiocholine; DTNB, 5,50 -dithio-bis(2-nitrobenzoic acid); 2-HQ, 2-hydroxyquinoline; HPBP, human phosphate binding protein. ⇑ Corresponding authors. Address: 503 Robert Grant Avenue, Walter Reed Army Institute of Research, Silver Spring, MD 20910, United States. Tel.: +1 301 319 9679/ 7307; fax: +1 301 319 9404. E-mail addresses: [email protected] (M. Valiyaveettil), [email protected] (M.P. Nambiar). 0887-2333/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.tiv.2011.02.012

olinergy, seizure, neuropathology and neurobehavioral deficits (Bajgar, 2004; Flynn and Wecker, 1986). The current treatment for OPs and CWNAs consist of a regimen of atropine, a muscarinic ACh receptor antagonist to counteract accumulation of ACh, 2-pralidoxime to reactivate OP-inhibited AChE, and benzodiazepines to treat seizures (Kadriu et al., 2009; Koplovitz and Stewart, 1994; Shih et al., 2007; Worek et al., 1996). There is no effective oxime against all nerve agents. Moreover, current therapies are not effective in preventing behavioral incapacitation and permanent brain damage due to OP/CWNA exposure. Pyridostigmine bromide, the approved pretreatment for soman does not pass the blood–brain barrier and protect central nervous system (Haigh et al., 2005). An alternative strategy for medical countermeasure against CWNA is the prophylactic treatment with biomolecules as scavengers for sequestration of OPs/CWNAs before they reach their physiological targets. Human butyrylcholinesterase (HuBChE) is the most promising scavenger for use as a pretreatment drug against nerve agents and a food and drug administration investigational new drug submission has been made for prophylactic treatment against CWNA toxicity (Doctor and Saxena, 2005; Lenz et al., 2005; Saxena

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et al., 2005; Sun et al., 2005). HuBChE is a very stable glycoprotein with a half life of 8–12 days when administered in vivo (Saxena et al., 2005; Sun et al., 2005). One of the limitation of this bioscavenger is its stoichiometric (1:1 ratio) nature of binding to CWNAs, requiring large amount of HuBChE (3 mg per mice or 60 mg/kg in guinea pigs) for in vivo protection against 2–5 LD50 nerve agent exposure (Doctor and Saxena, 2005; Lenz et al., 2005; Saxena et al., 2011, 2005). Researchers are now focused more on the development of enzymes functioning as catalytic bioscavengers which can effectively hydrolyze large amount of OPs and CWNAs (Lenz et al., 2007; Rochu et al., 2007; Stevens et al., 2008). One of the most promising proteins which can function as catalytic bioscavenger is paraoxonase 1 (PON1) (EC 3.1.8.1) (Costa et al., 2005b; Furlong et al., 2005; Josse and Masson, 2001; Lenz et al., 2007; Rochu et al., 2007; Stevens et al., 2008). It is a Ca2+dependent arylesterase glycoprotein with 355 amino acids (43– 45 kDa), expressed mainly in liver (Furlong et al., 1991; Hassett et al., 1991; Kuo and La Du, 1995; Primo-Parmo et al., 1996). PON1 exists in different polymorphic forms and the two common isoforms which have been comprehensively studied contain amino acid polymorphisms at position 55 and 192 (Costa et al., 2003a; La Du et al., 2001; Watson et al., 2001). The former influence the level of PON1 expression and the latter affects the activity of the enzyme. PON1 polymorphism in the promoter region reported to influence the level of protein expression (Brophy et al., 2001). Although the physiologic role of PON1 is still unclear, evidence exists for its protective effect against oxidative modifications of lipoproteins (Ayub et al., 1999; Costa et al., 2005a; Harel et al., 2004; Mackness et al., 2002). Other known functions of PON1 include metabolism of certain xenobiotics (Ginsberg et al., 2009). In humans, the paraoxonase gene family has three members, PON1, 2 and 3 with 65% amino acid similarity, but with different functions (Draganov et al., 2005; Primo-Parmo et al., 1996). PON1, the more abundant one has been studied in detail for the physiological functional roles. It has been also known that PON1 has lipolactonase activity similar to PON2 and 3 (Draganov et al., 2005; Khersonsky and Tawfik, 2005, 2006). Currently, there is much interest in developing PON1 as a broad spectrum catalytic bioscavenger to protect against OPs/CWNAs. PON1 is documented in literature as a potential catalytic bioscavenger due to its capacity to hydrolyze many OPs and nerve agents (Costa et al., 2005b; Furlong et al., 2005; Josse and Masson, 2001; Stevens et al., 2008). The protective role of PON1 in OP and CWNA toxicity and resistance has been initially derived from cross-species comparisons followed by animal experiments with purified PON1 in rodents and PON1 knockout mice (Furlong et al., 1998; Li et al., 1995; Shih et al., 1998). Earlier findings indicate that birds, which have very low plasma PON1 activity, are more sensitive to OP toxicity than rats, which in turn are more sensitive than rabbits, which have several-fold higher plasma PON1 activity (Costa et al., 2003b). Different levels of plasma PON1 activity has been implicated in Gulf War Syndrome (Hotopf et al., 2003). It has been also shown that administration of naked PON1 DNA lead to its expression and protection against soman (Fu et al., 2005). Human and rabbit serum PON1 has been purified earlier by several groups by different methods (Ekinci and Beydemir, 2010; Furlong et al., 1991, 1993; Gan et al., 1991; Rodrigo et al., 2001). Hydrolysis of different substrates by PON1 from specific sources has also been reported (Aharoni et al., 2004; Amitai et al., 2006; Billecke et al., 2000; Draganov et al., 2005; Harel et al., 2004; Josse et al., 2001; Li et al., 2000; Mackness et al., 1997; Masson et al., 1998; Otto et al., 2009; Richter et al., 2009; Watson et al., 2001; Yeung et al., 2004, 2005). A comparative analysis of the catalytic activity of these PON1s with human recombinant PON1 has not been evaluated so far. Moreover, direct spectrophotometric assays which have been used previously to demonstrate the catalytic effi-

ciency of PON1 require higher amount of substrates and enzymes with an uncertainty of complete hydrolysis of the substrates in the assays. We undertook a detailed and systematic study to compare the catalytic efficiency of purified human and rabbit serum and human recombinant PON1 against several substrates including OP insecticides using an in vitro assay system. The in vitro assay system is an indirect AChE back-titration assay that requires lower amounts of the substrates and enzyme with complete hydrolysis of the substrates. 2. Materials and methods 2.1. Materials Cibacron Blue 3GA agarose, DEAE-Sepharose, Sepharose CL-6B, Concanavalin A (Con A)-Sepharose, sodium deoxycholate, pnitrophenylacetate (p-NPA), AChE (Torpedo Californica), acetylthiocholine (ATCh), 5,50 -dithio-bis(2-nitrobenzoic acid) (DTNB), diisopropylfluorophosphate (DFP), diethyl paraoxon (DEP), dimethyl paraoxon (DMP), 2-hydroxyquinoline (2-HQ) and PON1 polyclonal antibody were purchased from Sigma (St. Louis, MO). SDS–PAGE running buffer, 4–20% Tris–glycine gradient gel, protein molecular weight marker, and SDS–PAGE transfer buffer were purchased from Invitrogen (Carlsbad, CA). Bicinchoninic acid (BCA) assay kit for protein estimation was purchased from Pierce (Rockford, IL). Immobilon polyvinylidene difluoride (PVDF) membrane and Centricon 30 microconcentrator were purchased from Millipore (Billerica, MA). Enhanced chemiluminescence (ECL) reagent for Western blot was purchased from GE Healthcare (Piscataway, NJ). 2.2. Purification of PON1 from human and rabbit serum Human serum handling and experiments were approved by a human exempt protocol through Division of Institutional Review Board of Walter Reed Army Institute of Research. Frozen human and rabbit serum were purchased from Innovative Research Inc. (Novi, MI). The purification of PON1 from human and rabbit serum was performed according to previously reported methodologies (Furlong et al., 1991; Gan et al., 1991; Rodrigo et al., 2001; Valiyaveettil et al., 2010, 2011a). Briefly, 100 ml human or rabbit serum was mixed with 300 ml of Cibacron Blue 3GA-agarose preequilibrated with buffer A (25 mM Tris/HCl, pH 8.0 containing 1 mM CaCl2, 5 lM EDTA and 3 M NaCl) at 4 °C overnight. The protein bound blue agarose was washed thrice with buffer A, transferred to a chromatography column, washed with excess buffer B (25 mM Tris/HCl, pH 8.0 containing 1 mM CaCl2 and 5 lM EDTA), followed by elution with a mild detergent (0.1% deoxycholate) in buffer B. Fractions of 10 ml were collected and aliquots were analyzed for PON1 activity using p-NPA as the substrate and the protein content was determined by BCA assay kit. The high PON1 activity containing fractions from blue agarose chromatography was pooled and diluted with equal volume of 50 mM Tris/HCl, pH 8.0 containing 10 mM CaCl2, 10 lM EDTA, 40% glycerol and 0.2% Triton X-100 and loaded to a DEAE-Sepharose ion-exchange column pre-equilibrated with buffer C (25 mM Tris/HCl, pH 8.0 containing 5 mM CaCl2, 5 lM EDTA, 20% glycerol and 0.1% Triton X-100). The column was washed with buffer C and bound proteins were eluted with a linear gradient of 0– 0.4 M NaCl in buffer C. Fractions of 10 ml were collected and assayed for PON1 activity and protein content as described above. The high PON1 activity containing fractions were pooled, dialyzed extensively with buffer C and applied to a second DEAE-Sepharose column followed by elution as described above. The high activity fractions were pooled, dialyzed, concentrated by using Centricon

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30 microconcentrator and applied to a Con A-Sepharose column pre-equilibrated with buffer D (25 mM Tris/HCl, pH 7.4 containing 1 mM CaCl2, 0.15 M NaCl and 0.1% Triton X-100). The bound enzyme was eluted with a linear gradient of 0–0.35 M a-methyl mannopyranoside in buffer D. Fractions (2.5 ml each) were collected and aliquots were analyzed for PON1 activity and protein content. The purified PON1 preparations were concentrated by using Centricon 30 microconcentrator, and 0.5 ml of purified enzyme was loaded onto a Sepharose CL-6B column pre-equilibrated with 20 mM Tris–HCl, pH 8.0 containing 150 mM NaCl and 1 mM CaCl2 and eluted with the same buffer. Fractions of 2 ml were collected and analyzed for PON1 activity and protein content. High PON1 activity fractions were pooled, dialyzed with 20 mM Tris–HCl, pH 8.0 containing 5% glycerol and 1 mM CaCl2 and further concentrated by using Centricon 30 microconcentrator. 2.3. Preparation of recombinant PON1 Human recombinant PON1 was expressed in Trichoplusia ni larvae (cabbage looper-worm) in collaboration with Chesapeake PERL (Savage, MD) and purified as described earlier (Otto et al., 2010; Valiyaveettil et al., 2010). Briefly, the larvae was orally infected by application of the viral inoculums (baculovirus expression vector containing human PON1 cDNA) on the surface of the diet followed by incubation at controlled conditions for 96 h, harvested and stored under frozen conditions until the recovery of PON1 enzyme. The recombinant protein was extracted and purified from the infected larvae by using classical protein purification technology including, ion-exchange and size-exclusion chromatography, ultra-filtration and finally by his-tagged affinity column (the baculovirus expression system has six histidine residues to facilitate this purification procedure). The purified recombinant PON1 showed >95% purity was stored under 80 °C until use. 2.4. SDS–PAGE and immunoblotting The purity of protein samples at each steps of PON1 purification from human and rabbit serum were analyzed by SDS–PAGE using XCell Sure Lock Mini-Cell electrophoresis system from Invitrogen (Carlsbad, CA). Briefly, 2–10 lg of protein samples were incubated with equal volume of 2 Laemmli sample buffer containing reducing agent at 100 °C for 5 min (Laemmli, 1970). The samples were loaded on 4–20% gradient Tris–glycine gels and run at a constant voltage of 100 V. The separated proteins were stained using Coomassie Blue stain and photographed with AlphaImager (Cell Biosciences, Santa Clara, CA). Purified human and rabbit PON1 and recombinant PON1 (0.1 lg each) resolved on Tris–glycine gels as described above were electroblotted onto PVDF membrane. The membrane was blocked with 2% non fat dry milk in phosphate buffered saline containing 0.1% Tween-20 (PBST) overnight at 4 °C, followed by incubation with PON1 polyclonal primary antibody (1:1000, Sigma, St. Louis, MO) for 2 h at room temperature. Membrane was washed thoroughly with PBST followed by incubation with peroxidase-labeled goat anti-rabbit secondary antibody (1:5000) for 1 h at room temperature, developed with ECL reagent and visualized using FluorChem HD2 system (Cell Biosciences, Santa Clara, CA). 2.5. PON1 enzyme activity and protein assay Aliquots of fractions collected during various purification steps as well as purified PON1 enzyme preparations were incubated with 1 mM of p-NPA substrate in 20 mM Tris–HCl buffer, pH 7.4 containing 1 mM CaCl2. The rate of formation of p-nitrophenol was measured at 405 nm using the Spectramax M5 Spectrophotometer (Molecular Devices, Sunnyvale, CA). Protein content in aliquots of

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fractions collected at various steps of purification was estimated by using BCA assay kit.

2.6. Enzyme kinetic assay Kinetic parameters were determined as follows. Equal amounts of purified human and rabbit serum PON1 and recombinant PON1 were incubated with p-NPA (concentration ranging from 0.1 to 4.0 mM in 20 mM Tris–HCl, pH 8.0 containing 1 mM CaCl2) or DEP (concentration ranging from 0.1 to 4.0 mM in 100 mM Tris– HCl, pH 8.0 containing 2 mM CaCl2) followed by the measurement of the formation of p-nitrophenol at 405 nm for 5 min using a spectrophotometer. The kinetic parameters, KM and Vmax, were determined graphically by using GraFit5 Version 5.0.13 software (Erithacus Software Ltd., Surrey, UK). Kcat values were expressed as the ratio of Vmax over moles of enzyme used in the reaction system. Enzyme activity in units was calculated from the initial linear rates of hydrolysis of substrates (p-NPA and DEP) using molar extinction coefficient of p-nitrophenol (18 mM1cm1).

2.7. AChE back-titration assay For the functional activity assay of PON1 against OPs, an in vitro back-titration of AChE Ellman assay was employed as described earlier (Ellman et al., 1961; Valiyaveettil et al., 2010; Wille et al., 2010). By using this methodology we can measure the complete hydrolysis of very low concentration OPs by PON1, compared to large concentrations of DEP and DMP used in the direct spectrophotometric method which produces high background yellow color in the reaction system. Briefly, different concentrations of various OPs (DFP, DEP or DMP) were incubated with 5 ng of AChE for 15 min at room temperature, followed by incubation with 1 mM of ATCh and 1 mM of DTNB. Activity was measured at 412 nm using Spectramax spectrophotometer. After determining the concentration of OPs required for 95% inhibition of AChE, different concentrations of purified human and rabbit serum PON1 and recombinant PON1 were pre-incubated with the specific concentration of OPs for 30 min at room temperature. The pre-incubated mixture was then incubated with 5 ng of AChE for another 15 min followed by the Ellman assay with ATCh and DTNB.

2.8. Inhibition of PON1 activity PON1 (5 lg) was pre-incubated with various concentrations of 2-HQ (10–500 lM) for 15 min at room temperature (Ahmad et al., 2010), followed by incubation with 2 mM p-NPA in 20 mM Tris HCl, pH 8.0 containing 1 mM CaCl2. The release of p-nitrophenol was measured at 405 nm using a spectrophotometer for 5 min. In another set of experiments, purified PON1 was pre-incubated with 2-HQ for 15 min, followed by incubation with DFP (10 lM) or DEP (1 lM) for 30 min. The reaction mixture was incubated with 5 ng of AChE followed by Ellman assay (Ellman et al., 1961). In the case of heat inactivated PON1 experiments, purified PON1 was kept at 100 °C for 10 min, followed by OP hydrolysis and Ellman assay as described above.

2.9. Data analysis All the assays were performed 3–5 times in triplicates. AChE back-titration results were expressed as % of control. Mean ± SEM values were plotted by using GraphPad Prism (version 4.03) software.

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3. Results 3.1. PON1 purification The methodology employed for the purification of PON1 was similar to previously described procedure, with modifications (Furlong et al., 1991; Gan et al., 1991; Rodrigo et al., 2001; Valiyaveettil et al., 2010, 2011a). The chromatographic profile of PON1 purification from human serum is shown in Fig. 1. The elution profiles of rabbit serum PON1 was very similar to human serum PON1 (data not shown). Purified human and rabbit serum PON1 after the gel filtration showed a specific activity of 13–15 units/mg protein with 95–98-fold purification, respectively. Purified human recombinant PON1 expressed in T. ni larvae is PON1 allozyme A (amino acid Glu at position 192) and showed a specific activity of 8.5 units/mg protein as reported earlier (Valiyaveettil et al., 2010). SDS–PAGE analysis of the PON1 preparations at different steps of the purification procedure is shown in Fig. 2A. Both human and rabbit serum purified PON1 samples showed protein bands similar to the size of purified human recombinant PON1 after blue agarose and DEAE-Sepharose chromatography with significant amount of albumin and apolipoprotein AI (Apo AI) as the major impurities (lanes 1 and 2 in Fig. 2A). The Con A-Sepharose affinity purification eliminated majority of the impurities in the PON1 preparation, though minor amounts of Apo AI, albumin and Con A which leached out from the affinity column were still present

(lanes 3 and 4 in Fig. 2A). Human serum purified PON1 showed a protein band in the probable region (39 kDa) of human phosphate binding protein (HPBP) after DEAE-Sepharose chromatography, which got really diminished after Con A-Sepharose affinity chromatography. Purified PON1 from gel filtration chromatography showed high purity in SDS–PAGE (lanes 5 and 6 in Fig. 2A). Human serum purified PON1 showed mainly a single protein band at 45 kDa, while rabbit serum purified PON1 showed three bands in the 40–45 kDa molecular weight regions similar to purified recombinant human PON1 which showed three bands at 42– 45 kDa range (lanes 5, 6 and 7 in Fig. 2A). The purity of PON1 sample was further evaluated by Western blotting with a specific polyclonal antibody against PON1. The purified PON1 preparations from both human and rabbit serum cross reacted with the PON1 polyclonal antibody as shown in Fig. 2B. As observed with SDS–PAGE analysis, human serum PON1 showed cross reactivity to the upper band (45 kDa), while both rabbit serum PON1 and human recombinant PON1 cross-reacted mainly with the two upper bands with minor cross reactivity with the lower band (Fig. 2B). 3.2. Hydrolysis of substrates by purified PON1 The kinetic analysis of purified human and rabbit serum PON1 and recombinant PON1 with p-NPA and DEP is shown in Fig. 2C and D, respectively. The catalytic efficiency of purified human

Fig. 1. Chromatographic profiles of PON1 purification. (A) PON1 from human and rabbit serum was first purified using Cibacron Blue-Agarose affinity column followed by elution with 0.1% deoxycholate buffer. (B) The high activity enzyme fractions from (A) were pooled and purified by using DEAE-Sepharose ion-exchange column and the enzyme was eluted with 0–0.4 M NaCl gradient. (C) High activity fractions from (B) were pooled and applied to a Con A-Sepharose column followed by washing with the buffer as described in Section 2, and the bound enzyme was eluted with 0–0.35 M a-methyl mannopyranoside gradient. (D) The dialyzed pooled PON1 fraction from (C) was concentrated by using Centricon ultrafiltration and applied to Sepharose CL-6B gel filtration column for final purification. In each step PON1 activity of the fractions (open circle) was measured by using p-NPA as the substrate and the protein content (closed circle) was analyzed by BCA assay kit. Shown are the representative figures for purification of PON1 from human serum.

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Fig. 2. Characterization of purified PON1. (A) SDS–PAGE analysis of PON1 preparations. Lanes 1 and 2 are after DEAE-Sepharose chromatography; lanes 3 and 4 are after Con A-Sepharose chromatography; and lanes 5 and 6 are after gel filtration chromatography. Lanes 1, 3 and 5-human PON1 (HPON1); lanes 2, 4 and 6-rabbit PON1 (RPON1); and lane 7-recombinant PON1 (rePON1). (B) Western blot analysis of purified PON1 preparations. Lane 1, HPON1; lane 2, RPON1; lane 3, rePON1. In panels A and B, the molecular weight markers are shown on the left side. (C) Purified PON1 (0.35 lg) was incubated with varying concentrations (0.05–4.0 mM) of p-NPA and the release of p-nitrophenol was measured at 405 nm. (D) Purified PON1 (2 lg) was incubated with varying concentrations (0.05–4.0 mM) of diethyl paraoxon and the release of p-nitrophenol was measured at 405 nm. In panels C and D, closed circle – HPON1, closed square – RPON1 and closed triangle – rePON1.

serum PON1 (4.7  106) and human recombinant PON1 (4.2  106) were similar and significantly lower than that of rabbit serum purified PON1 (7.5  106) against p-NPA substrate (Fig. 2C). The catalytic efficiency of purified PON1 was significantly lower with DEP than that compared with p-NPA substrate (compare Fig. 2D with C). Purified human serum PON1 and recombinant PON1 showed similar catalytic efficiency (7.3  103 and 6.5  103, respectively), significantly lower than that of purified rabbit serum PON1 (1.8  104) against DEP (Fig. 2D). 3.3. Organophosphate hydrolysis by purified PON1 To test the hydrolysis of OPs by purified PON1, we employed an in vitro AChE back-titration assay method with the classical Ellman reaction as reported earlier (Valiyaveettil et al., 2010). We tested the hydrolyzing potential of DFP, DEP and DMP by multiple PON1s using the AChE back-titration assay. First, we determined the optimum level of DFP, DEP or DMP required for 95% inhibition of AChE in Ellman assay. As shown in Fig. 3A, a concentration in the range

of 10 lM of DFP, or 1.0 lM of DEP, or 2.0 lM of DMP resulted in approximately 95% inhibition of AChE enzyme activity in the reaction system. In the next step, varying concentrations of purified PON1s were pre-incubated with 10 lM DFP and AChE was added to the hydrolyzed or non-hydrolyzed mixture followed by the Ellman assay. All three sources of purified PON1s hydrolyzed DFP as indicated by no inhibition of AChE and higher values by Ellman assay (Fig. 3B). The amount of PON1 enzyme required for 100% hydrolysis of DFP was 20 lg for purified human serum PON1, 5 lg for purified rabbit serum PON1 and 7 lg for recombinant PON1. These results demonstrate that rabbit serum PON1 hydrolyzes DFP much more effectively than purified human serum PON1 and recombinant PON1. The catalytic activity of purified PON1 was also evaluated by using another OP, diethyl paraoxon as described above. DEP showed 95% inhibition of AChE activity at a concentration of 1 lM in Ellman assay (Fig. 3A). This concentration of DEP was completely hydrolyzed in the presence of human and rabbit serum purified PON1 and recombinant PON1 (Fig. 3C). Purified rabbit

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Fig. 3. Hydrolysis of OPs with PON1. (A) Indicated concentrations of DFP, DEP or DMP were pre-incubated with 5 ng of AChE for 15 min, followed by incubation with 1 mM of ATCh and DTNB and the absorbance was measured at 412 nm for 5 min in a kinetic mode using SpectraMax spectrophotometer. (B–D) Indicated concentrations of rePON1, RPON1 or HPON1 were incubated with 10 lM of DFP (B), 1 lM of DEP (C), or 2 lM of DMP (D) for 30 min followed by incubation with 5 ng of AChE for 15 min. The samples were further incubated with 1 mM of ATCh and DTNB and the reaction was monitored at 412 nm for 5 min in a kinetic mode.

serum PON1 showed higher catalytic activity with DEP, requiring only 1 lg of the enzyme for complete hydrolysis. In the case of human serum purified PON1, 5 lg of enzyme could hydrolyze 100% of the substrate, where as 8 lg of purified human recombinant PON1 was required for the same effect. Catalytic activity of purified PON1 with DMP (compound structurally related to DEP) is also shown in Fig. 3D. The concentration of DMP required for 95% inhibition of AChE activity was approximately 2 lM (Fig. 3A). High concentration of purified human serum PON1 (75 lg) and rabbit serum PON1 (25 lg) was required to hydrolyze 2 lM concentration of DMP (Fig. 3D). Recombinant human PON1 could not hydrolyze DMP even at higher concentrations used (data not shown). A significant difference in the catalytic activity was observed between purified human and rabbit serum PON1 and recombinant PON1 with DMP. 3.4. Specificity of PON1 catalysis To exclude the possibility of any OP and substrate hydrolyzing activity due to minor contaminants in the purified PON1, purified enzyme was inhibited with 2-HQ, specific inhibitor of PON1. Incubation of PON1 with 2-HQ showed a dose-dependent inhibition of enzyme activity against p-NPA substrate, while incubation of AChE with 2-HQ did not show any inhibitory activity as reported earlier (Valiyaveettil et al., 2010). These results were reproducible with purified human and rabbit serum PON1 as well as recombinant PON1 (data not shown). Inclusion of 2-HQ in the AChE back titration assay system with DFP and DEP abolished the OP hydrolyzing property of PON1 (Fig. 4A). Furthermore, heat inactivation of PON1 used in the reaction system also abolished the OP hydrolysis char-

acter of PON1 (Fig. 4A). These data confirm the specificity of PON1 activity in the enzyme preparations used. To ascertain whether purified PON1 acts as a catalytic bioscavenger, the AChE back-titration assay performed after incubation of purified PON1 with 3-fold higher concentration of OPs. Single dose of PON1 could efficiently hydrolyze larger amount of DFP and DEP (Fig. 4A). The activity of PON1 measured by p-NPA was not affected after incubation with OPs (Fig. 4B). These results indicate that PON1 acts as a catalytic bioscavenger.

4. Discussion The results presented in this paper provide a comparative evaluation of purified human and rabbit serum and human recombinant PON1 against several substrates including OP insecticides using an in vitro AChE back-titration assay system. We purified PON1 enzymes from human and rabbit serum to homogeneity and the human recombinant PON1 was expressed and purified from T. ni larvae. Although PON1 has been purified earlier, the modified purification method yielded more pure PON1 with catalytic activity comparable to previously reported ones (Aharoni et al., 2004; Amitai et al., 2006; Harel et al., 2004; Josse et al., 2001; Masson et al., 1998; Otto et al., 2009; Rochu et al., 2007; Yeung et al., 2004). Purified rabbit serum and human recombinant PON1 showed multiple bands in SDS–PAGE and immunoblotting (Fig. 2A and B) owing to different levels of glycosylation of the enzyme in different systems as reported earlier (Gan et al., 1991; Otto et al., 2010). Multiple bands in purified recombinant PON1 have also reported

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Fig. 4. The specificity of PON1 enzyme activity and the efficiency of PON1 as a catalytic bioscavenger. (A) RPON1 (1 or 5 lg) was pre-incubated with or without 2-HQ (0.5 or 3 mM) for 15 min, followed by incubation with DFP (10 lM) or EP (1 lM) for 30 min and the AChE back-titration assay was performed. The same experiments were performed by using heat-inactivated RPON1 without using 2-HQ. The experiments were also done by using higher concentrations of DFP (30 lM) and EP (3 lM). (B) PON1 enzyme activity in the presence of OPs. RPON1 (1 or 5 lg) was pre-incubated with DFP (10 or 30 lM), and EP (1 or 3 lM) for 30 min, followed by the PON1 activity assay using p-NPA substrate as described in Section 2.

previously (Otto et al., 2010). Escherichia coli expressed and purified human serum PON1 showed single band with high catalytic activity indicating that glycosylation may not be critical for PON1 activity (Gan et al., 1991; Stevens et al., 2008). Also the E. coli expressed PON1 protected mice against OPs (Stevens et al., 2008). It has been reported that HPBP co-purifies with the PON1 from human serum which is suggested as a stabilizing partner (Morales et al., 2006; Renault et al., 2006; Rochu et al., 2007). In contrast, other reports suggest the lack of co-purification of HPBP with human serum purified PON1 (Liu et al., 2008). In addition, recombinant human PON1 that does not has HPBP showed high catalytic efficiency with multiple bands indicating that PON1 has oligomeric forms as described earlier (Josse et al., 2002). In our purification procedure a protein corresponding to HPBP was co-purified in the initial stages of purification up to DEAE-Sepharose chromatography along with albumin and Apo-AI which got disappeared in the later stages of purification (see Fig. 2A). The purified PON1 enzyme devoid of HPBP was able to hydrolyze multiple substrates including OPs (Figs. 2 and 3). The stability of PON1 enzyme without HPBP may be at risk (Rochu et al., 2007), but in our studies storage of PON1 with 3% glycerol maintained the activity of the enzyme for at least few months. The purified PON1 showed significant catalytic activity against multiple substrates. The catalytic activity of rabbit serum PON1 is much higher than human serum purified and recombinant PON1, while the catalytic efficiency of human serum purified and recombinant PON1 were similar. Even though rabbit serum PON1 has 85% amino acid identity to human serum PON1, it shows higher levels of PON1 enzyme activity mainly due to amino acid residue Lys at position 192 (Aharoni et al., 2004; Furlong et al., 1991; Kuo and La Du, 1995; Watson et al., 2001). Purified rabbit serum PON1 was more efficient than that of human recombinant PON1 and human serum PON1 in hydrolyzing DFP, DEP and DMP. These results clearly demonstrate that there are significant differences in the hydrolyzing efficiency of PON1 against different substrates. Recently, we reported the efficient hydrolysis of CWNA tabun by purified human and rabbit serum and human recombinant PON1 (Valiyaveettil et al., 2010). The hydrolyzing potential of purified PON1 with tabun was significantly higher than that observed with sarin and soman, which clearly reflects the difference in the catalytic efficiency of multiple PON1s against different substrates

as reported in the present study. It is intriguing to note that purified human and rabbit serum and human recombinant PON1 hydrolyzed large concentration of DFP (10 lM) compared to DEP or DMP (1 or 2 lM) showing higher catalytic efficiency towards DFP. The protective efficacy of purified PON1 against CWNAs sarin and soman has been evaluated using a microinstillation inhalation exposure model developed in our laboratory (Nambiar et al., 2006; Valiyaveettil et al., 2011a,b). In summary, in vitro AChE back-titration assay of purified PON1 preparations from multiple sources showed hydrolysis of several substrates. The catalytic activity of the enzyme was different with regard to substrate and the types of PON1. The activity of PON1 towards OP was specific to the enzyme since inhibition of PON1 completely abolished the OP hydrolysis potential. PON1 could hydrolyze larger doses of OPs by preserving the intrinsic activity of the enzyme. These results emphasize the importance of detailed analysis of the catalytic activity of new molecular forms of PON1 that are currently being developed to increase its catalytic efficiency for protection against CWNAs and OP insecticides poisoning. Disclosure The opinions and assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Army, the Navy, or the Department of Defense, USA. Conflict of interest statement All authors have no financial or personal conflict of interest. Acknowledgement This work was supported by funding from Defense Threat Reduction Agency (DTRA) Grant #1.D0017_08_WR_C. References Aharoni, A., Gaidukov, L., Yagur, S., Toker, L., Silman, I., Tawfik, D.S., 2004. Directed evolution of mammalian paraoxonases PON1 and PON3 for bacterial expression and catalytic specialization. Proc. Natl. Acad. Sci. USA 101, 482–487.

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