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Specific MPO inhibitors, salicylhydroxamic acid or 4-aminobenzoic acid hydrazide, are added to mea- sure the activity of other heme-containing peroxidases ...
ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2009, Vol. 35, No. 5, pp. 566–575. © Pleiades Publishing, Ltd., 2009. Original Russian Text © I.V. Gorudko, O.S. Tcherkalina, A.V. Sokolov, M.O. Pulina, E.T. Zakharova, V.B. Vasilyev, S.N. Cherenkevich, O.M. Panasenko, 2009, published in Bioorganicheskaya Khimiya, 2009, Vol. 35, No. 5, pp. 629–639.

New Approaches to the Measurement of the Concentration and Peroxidase Activity of Myeloperoxidase in Human Blood Plasma I. V. Gorudkoa,1, O. S. Tcherkalinab, A. V. Sokolovb, M. O. Pulinab, E. T. Zakharovab, V. B. Vasilyevb, S. N. Cherenkevicha, and O. M. Panasenkoc a

Department of Biophysics, Belarusian State University, pr. Nezavisimosti 4, Minsk, 220030 Belarus b Institute for Experimental Medicine, Russian Academy of Medical Sciences, ul. Akademika Pavlova 12, St. Petersburg, 197376 Russia c Research Institute of Physico-Chemical Medicine, ul. Malaya Pirogovskaya 1a, Moscow, 119992 Russia Received March 2, 2009; in final form, March 20, 2009

Abstract—A novel method for spectrophotometrical measurement of myeloperoxidase (MPO) activity in plasma with o-dianisidine (DA) as a substrate is proposed. We have determined the optimal conditions, including the pH and hydrogen peroxide concentration, under which MPO is the main contributor to DA oxidation in plasma. Specific MPO inhibitors, salicylhydroxamic acid or 4-aminobenzoic acid hydrazide, are added to measure the activity of other heme-containing peroxidases (mainly hemoglobin and its derivatives) and subtract their contribution from the total plasma peroxidase activity. Plasma MPO concentrations are quantified by a new enzyme-linked immunosorbent assay (ELISA) developed by us and based on the use of antibodies raised in rats and rabbits. The sensitivity of this ELISA is high: 0.2–250 ng/ml. A direct and significant (P < 0.0001) correlation was observed between the MPO activities measured spectrophotometrically and the MPO level determined by ELISA in blood samples from 38 healthy donors. The proposed approaches to MPO measurement in plasma can be used to evaluate the enzyme activity and concentration, as well as the efficacy of mechanisms by which MPO is regulated under physiological conditions and against the background of various inflammatory diseases. Key words: myeloperoxidase, plasma peroxidase activity, blood plasma, o-dianisidine, hemoglobin, enzymelinked immunosorbent assay. DOI: 10.1134/S1068162009050057

INTRODUCTION Myeloperoxidase (MPO2; donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7) is a glycoprotein. It consists of two identical dimers linked via a disulfide bond. Each dimer contains a 57-kDa glycosylated α subunit covalently attached to heme (protoporphyrin IX coordinated with Fe3+) and a 12-kDa unglycosylated β subunit [1]. This enzyme is located mainly in azurophilic granules of neutrophils, where it constitutes up to 5% of the total protein [2], and in monocytes (0.9%) [3]. A schematic diagram of MPO action is shown in Fig. 1. Hydrogen peroxide is formed in vivo by a respiratory explosion. It converts MPO released from activated neutrophils to compound I (Fig. 1, reaction 1). This MPO form has a high redox potential. It can be involved in two processes. One of them is the 2-electron 1

Corresponding author; phone: +375 (17) 209 5437; fax: +375 (17) 209 5445; e-mail: [email protected] 2 Abbreviations: MPO, myeloperoxidase; DA, o-dianisidine.

oxidation of halogenides (Cl–, Br–, and I–) and pseudohalogenides (SCN–). In this process, highly reactive hypohalogenous acids (HOX, where ï– is a halogenide or pseudohalogenide) form, the so-called halogenation cycle is closed, and the enzyme returns to the native MPO–Fe3+ form (Fig. 1, reactions 1 and 2). In the other process, compound I is first converted into compound II and then to the native enzyme by sequential 1-electron oxidation of various peroxidase substrates (AH in Fig. 1) to close the peroxidase cycle (Fig. 1, reactions 1, 3, and 4). Thus, the enzyme possesses both halogenation and peroxidase activities [4, 5]. Thus, MPO generates highly reactive oxidative species: HOCl, HOBr, free radicals, etc. They mediate the main antimicrobial function of neutrophils [4]. This role of MPO is confirmed by the fact that MPO-deficient patients are generally more susceptible to infections [6]. The biological action of MPO is largely determined by the balance between its secretion to the extracellular

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Fig. 1. Major reactions of the halogenation and peroxidase cycles catalyzed by MPO. X−, halogenide or pseudohalogenide (SCN−); AH, peroxidase substrate.

space at the stage of neutrophil degranulation, on one hand, and its inactivation and utilization in the tissue, as well as degradation of oxidative species produced in MPO reactions, on the other hand. The enzyme can act detrimentally as a result of neutrophil secretory degranulation or death [4]. In this case, strong oxidative species are produced by MPO induce lipid peroxidation and modify proteins and nucleic acids by halogenation, nitration, oxidation, and linking, thereby damaging host tissues in inflammatory foci. As MPO is a biochemical marker of neutrophil activation, it can play a decisive role in atherosclerosis [7], cardiovascular diseases [8], cancer [9], neurodegenerative diseases [10], lung respiratory dysfunction [11], kidney disorders [12], systemic vasculitides [13], rheumatoid arthritides [14], etc. Neutrophil degranulation or lysis release significant amounts (up to 1.11 µg/ml, or 7.78 nM) of MPO into blood [8]. Therefore, monitoring of the enzyme concentration is essential. In a hospital, MPO analysis is carried out by ELISA with monoclonal antibodies [7, 8, 15]. This method is sensitive and specific. Unfortunately, it cannot evaluate the enzyme activity [15], which actually determines the involvement of MPO in the development of socially significant diseases. The enzyme activity can be assessed via the production of hypohalogenous acids (halogenation activity) [5, 6] or via substrate oxidation in the peroxidase cycle (peroxidase activity) [5, 17]. However, the rates of HOCl and HOBr reactions with functional groups of blood plasma biomolecules are so high (k > 106 M–1s–1) [18] that present day methods cannot record the formation of hypohalogenous acids in blood serum or, as a consequence, determine the halogenation activity. This activity can be measured only in the purified enzyme or, at least, in neutrophil suspensions [16]. RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

The assessment of MPO peroxidase activity is hampered by the fact that blood plasma contains hemeassociated proteins operating as peroxidases, mainly hemoglobin and its derivatives [19]. Even slight hemolysis increases the hemoglobin concentration in plasma to micromoles. Moreover, some proteins, e.g., ceruloplasmin, display oxidase activity by oxidizing aromatic substrates without hydrogen peroxide [20]. It should be taken into account that blood contains factors regulating MPO activity. In particular, ceruloplasmin forms complexes with MPO and inhibits its activity [21]. All of these circumstances should be taken into account to determine the actual MPO activity in blood plasma. The simultaneous measurement of the MPO concentration and its activity would provide data on the MPO concentration and activity in plasma and, in addition, allow for an assessment of the efficiency of systems regulating MPO activity in blood. Here, we propose a spectrophotometrical method for measuring MPO peroxidase activity directly in blood plasma and improve the ELISA for determining MPO concentration. RESULTS AND DISCUSSION Spectrophotometrical method. Four commonly known aromatic substrates were tested to develop a convenient system for detecting MPO peroxidase activity: guaiacol, 4-chloro-1-naphthol, o-dianisidine (DA), and tyrosine. Fig. 2 shows that MPO activity increases in a dose-dependent manner with all the substrates in the model buffer system with hydrogen peroxide. However, corresponding experiments with blood plasma and exogenous MPO showed peroxidase activity only in the reaction with DA (Fig. 3, curve 3). The addition of plasma with MPO to the reaction mixture with guai-

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acol, tyrosine, or 4-chloro-1-naphthol did not initiate the enzymatic reaction (data not shown). No absorbance increase was observed with the absence of hydrogen peroxide either (Fig. 3, curve 1). The addition of H2O2 to plasma was accompanied by a small but statistically significant absorbance increase (Fig. 3, curve 2) owing to DA oxidation just in the peroxidase reaction. As mentioned above, the overall peroxidase activity of plasma is contributed, in addition to MPO, by hemoglobin and other heme-associated proteins utilizing H2O2 [19]. Indeed, the addition of 1 µM hemoglobin to plasma resulted in a significant A460 increase (Fig. 3, curve 4). Apparently, hemoglobin adds much to the overall peroxidase activity under the experimental conditions (1 mM ç2é2, pH 7.0). Thus, MPO peroxidase activity was detected in plasma only with DA as a substrate. However, DA is not MPO specific, and there are other proteins that contribute to DA conversion and hamper the determination of true MPO peroxidase activity. This fact poses two problems. First, conditions should be chosen under

which MPO would make the largest contribution to DA oxidation by plasma; second, the effect of side peroxidase activity should be eliminated; that is, the contribution of hemoglobin to DA oxidation should be minimized. It is known that MPO activity depends greatly on the ç2é2 concentration [22, 23]. The ç2é2 concentration dependences of the peroxidase activity of plasma supplemented with MPO or hemoglobin are shown in Fig. 4a. For MPO, the curve has a maximum at 100 µM. At higher ç2é2 concentrations, MPO activity decreases dramatically. This observation is in agreement with earlier data [22, 24]. With the presence of hemoglobin, plasma peroxidase activity increases linearly with the ç2é2 concentration, at least up to 1 mM. The contribution of hemoglobin peroxidase activity at 100 µM is negligible. Thus, the optimum ç2é2 concentration for measuring MPO peroxidase activity in plasma is 100 µM. The optimum pH for MPO varies within 4.5–6.0 [16, 24]. It depends on various factors: ionic strength, substrate species and concentration, and presence of

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other compounds [23]. The MPO action on DA can be modulated by ceruloplasmin [21] or blood lipoproteins [25]. For this reason, we compared the pH dependences of MPO activity in the buffer solution with and without plasma (Fig. 4b). The results indicate that plasma shifts the optimum pH range in the acidic direction (4.5) in comparison with the buffer solution (5.5–6.0). We suppose that this change is associated with the ceruloplasmin present in plasma. It interacts with MPO and inhibits its activity with regard to DA [21]. The MPO–ceruloplasmin complex dissociates completely at pH < 3.9 [26]. Therefore, MPO activity increases under acidic conditions, not being inhibited by ceruloplasmin. Note that the peroxidase activity of hemoglobin with the presence of plasma decreases at pH 4.5. The rates of DA oxidation by 1 µM of hemoglobin without and with plasma are 0.06 and 0.04 ∆A460/min, respectively. Thus, the minimum contribution of heme-associated proteins to DA oxidation by plasma is reached at 100 µM ç2é2 and pH 4.5. Typical kinetic curves for DA oxidation at various concentrations of exogenous MPO and hemoglobin under conditions chosen in our study are shown in Figs. 5a and 5b, respectively. An elevated A460 value in the plasma + DA + ç2é2 mixture is clearly recorded even at minor MPO amounts: 50 ng/ml or 0.34 nM (Fig. 5a, curve 3). Hemoglobin shows a notable absorbance increase only at a much higher concentration: 0.5 µM (Fig. 5b, curve 3). The concentration dependences of MPO and hemoglobin activities calculated from kinetic curves with subtraction of the intrinsic peroxidase activity of plasma (Fig. 5, curve 2) are shown in insets. It is apparent that MPO and hemogloRUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

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bin activities linearly increase with concentration. Thus, in spite of the fact that the chosen conditions minimize the effect of contaminating hemoglobin in MPO assays in plasma, its contribution to recorded peroxidase activity values cannot be entirely eliminated, particularly, when its concentration reaches micromoles owing to hemolysis. Experiments on the elimination of hemoglobin contribution to MPO activity were repeatedly described by other authors. Gel filtration was applied to measure MPO activity in a tissue sample [27]. 4-Aminobenzoic acid hydrazide, an MPO inhibitor, was used for selective determination of MPO activity in synovial fluid [28]. To measure the hemoglobin contribution, we also added specific MPO inhibitors: 4-aminobenzoic acid hydrazide [29] and salicylhydroxamic acid [30]. Each inhibitor was added to plasma with various MPO con-

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Fig. 5. Kinetic curves for oxidation of 380 µM DA with 100 µM H2O2 and blood plasma 1 : 13.3 v/v in a phosphate citrate buffer, pH 4.5, with the presence of exogenous (a) MPO or (b) hemoglobin: 1, Oxidation by plasma without MPO, hemoglobin, or H2O2 (oxidase activity); 2–6, concentrations of (a) MPO 0, 50, 75, 100, and 125 ng/ml, and (b) hemoglobin 0, 0.5, 1, 1.5, 2 µM, respectively. Insets show concentration dependences of the peroxidase activities of (a) MPO and (b) hemoglobin calculated from the kinetic curves minus the intrinsic peroxidase activity of plasma (curve 2).

centrations at a constant hemoglobin concentration or various hemoglobin concentrations at a constant MPO concentration. Peroxidase activity was recorded in all plasma samples under the conditions chosen in our study. The results are shown in Fig. 6. Only hemoglobin-associated peroxidase activity was detected with the presence of MPO inhibitors. These data prove that the inhibitors chosen specifically inhibit MPO without affecting hemoglobin activity. In other words, they allow the contribution of MPO to plasma peroxidase activity to be eliminated and the hemoglobin contribution to be determined. To summarize, our MPO assay in blood plasma involves the spectrophotometrical measurement of the rate of DA oxidation at pH 4.5 and 100 µM H2O2 with and without an MPO inhibitor: 100 µM salicylhydroxamic acid or 50 µM 4-aminobenzoic acid hydrazide. The peroxidase activity of plasma MPO is determined as the difference between the total peroxidase activity, measured with H2O2 but without any inhibitor, and the peroxidase activity measured with the presence of H2O2 and an MPO inhibitor. It should be noted that plasma possesses oxidase activity and catalyzes DA oxidation even without hydrogen peroxide. However, our method eliminates the contribution of plasma oxidase activity as well. The development of highly sensitive ELISA. We developed a sandwich ELISA with polyclonal antibodies raised in various animal species to determine the total MPO content in plasma. The protocol is outlined in Table 1. In our pilot experiments, we determined the optimum conditions for all analysis steps. The optimum concentration of primary affinity rat antibodies for

immobilization in polystyrene plates in a sodium carbonate buffer, pH 9.4, was 5 5 µg/ml. Lower concentrations decreased the final signal, probably because of incomplete MPO sorption, and higher concentrations caused excessive (loose) antibody sorption and the desorption of antibody–MPO complexes from the plate. Albumin, gelatin, and milk powder proteins were used for blocking. With milk powder proteins, the linear dependence of A492 on lg[MPO] was observed in the widest range, probably owing to the least effect of the proteins on MPO–antibody binding. The effect of plasma components on MPO sorption was eliminated by the addition of 0.05% Tween 20 at all ELISA steps. Measurements with twofold serial dilutions of 200 or 250 ng/ml stock MPO solutions revealed a linear correlation between A492 and lg[MPO] within the range 3–250 ng/ml (Fig. 7). It should be mentioned though, that the threshold sensitivity was even better: The A492 value for the well with 0.2 ng/ml MPO differed significantly from the control well (ê < 0.001). The difference between replications in one experiment was always within 2%. The differences between independent experiments reached 6%, probably owing to differences in the antibody sorption during the first step of analysis. For this reason, calibration against standard MPO samples are required in each experiment. ELISA protocols where sensitivity was improved by using antibodies from various animal species were described in [31]. Our protocol differs in that we used polyclonal rather than monoclonal antibodies from rats and rabbits. The avidity of polyclonal antibodies is enhanced by the presence of several paratopes, whereas the difference between epitopes for rat and rabbit anti-

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Fig. 6. Effect of 100 µM salicylhydroxamic acid and 50 µM (4-aminobenzoyl)hydrazide on the peroxidase activity of blood plasma (1 : 13.3 v/v in a phosphate citrate buffer, pH 4.5, 380 µM DA, and 100 µM H2O2) containing (a) 1 µM hemoglobin and various MPO concentrations or (b) 100 ng/ml MPO and various hemoglobin concentrations.

bodies diminishes the probability of their competition for the antigen. Indeed, the resulting threshold sensitivity of our protocol proved to be better than with monoclonal antibodies: 0.2 vs. 15 ng MPO/ml [31]. The wide linear range of the method, 3–250 ng/ml, allows for the detection of both normal and pathologically elevated MPO concentrations. Comparison of methods for measurement of MPO. Features of ELISA and the spectrophotometrical method are summarized in Table 2. Both methods are applicable in wide concentration ranges, i.e., they can detect both normal and pathological MPO concentrations in plasma. The threshold sensitivity of ELISA is better than that of the spectrophotometrical method, but the former demands calibration in each experiment. The fastness of the spectrophotometrical method is an undeniable advantage. Moreover, it requires no sophisticated equipment; therefore, it is convenient for ordinary hospitals. We compared the results of spectrophotometrical and ELISA analyses of blood plasma samples from 38 donors. The correlation between the MPO concenRUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

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Fig. 7. Typical standard log chart for the MPO concentration (ng/ml) dependence of A492 for the calculation of MPO concentration in plasma in ELISA: R2 = 0.9958; y = 0.75x – 0.1819.

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tration and peroxidase activity in plasma is shown in Fig. 8. Statistically valid correlations (P < 0.0001) between MPO activity and concentration were found with both salicylhydroxamic acid (Fig. 8a) and 4-aminobenzoic acid hydrazide (Fig. 8b), the correlation coefficients being 0.694 and 0.701, respectively. The proximity of these values and the high linear correlation coefficient (0.903) between MPO peroxidase activities measured with these inhibitors (Fig. 8c) indicate that MPO peroxidase activity in plasma can be accurately determined with either inhibitor. One of the methods for the assessment of MPO activity in plasma is the record of concentrations of socalled MPO biomarkers. They are substances formed in reactions between oxidizers produced by MPO and functional groups of biomolecules. Immunohistochemical and mass-spectrophotometrical studies have demonstrated the presence of MPO and products of MPOmediated reactions in atherosclerotic plaques, but not in the intact intima [32]. The products included chlorinated and nitrated tyrosine residues and chlorinated lipids. Elevated concentrations of products of MPO-mediated halogenation, 3-chlorotyrosine-containing proteins, were detected in the bronchoalveolar lavage fluid from lung patients [11] and in brain tissues of Alzheimer’s disease patients [33]. Proteins modified by HOCl were immunohistochemically detected in glomerulonephritic kidneys [34]. However, all of these methods are complex and labor consuming. They demand expensive sophisticated equipment. As for blood plasma analysis, the spectrophotometrical methods described thus far determine only the total peroxidase activity [35]. An attempt was made to assess MPO activity in blood serum by the common spectrophotometrical method applied to solutions of the isolated enzyme [36]. In that study, DA was used as

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Table 1. ELISA for MPO in plasma No. 1 2 3 4

5 6 7 8 9

Step

Reagent, quantity per well, conditions, and duration

5 µg/ml rat antibodies against MPO in a 0.1 M sodium carbonate buffer, pH 9.4, 100 µl, +4°C, 17 h Washing 0.05% v/v Tween 20 in PBS, 200 µl, +20°C, 3 × 30 s Blocking BLOTTO-T, 200 µl, +37°C, 1 h Standards/samples Same as step 2. Standard solutions were prepared by serial twofold MPO dilutions in BLOTTO-T, concentrations within 3–200 ng/ml. Samples were prepared by the dilution of plasma samples in BLOTTO-T by factors of 4–256, 100 µl, +37°C, 1 h Rabbit IgG antibodies Same as step 2; 10 µg/ml rabbit antibodies against MPO in BLOTTO-T, 100 µl, +37°C, 1 h Labeled antibodies Same as step 2; goat antibodies against rabbit IgG antibodies conjugated with horseradish peroxidase (1 : 5000, BioRad) in BLOTTO-T, 100 µl, +37°C, 1 h Substrate Same as step 2; 11 ml of a 0.1-M sodium citrate buffer pH 4.0 and 5 mM H2O2 are added to 10 mg of o-phenylenediamine in 1 ml of ethanol; 100 µl, +37°C, 4–5 min Termination 50 µl of 6 M H2SO4, 30 s Measurement Microplate reader, A492 Rat IgG antibodies

Table 2. Comparison of the spectrophotometrical method (SM) and ELISA for analysis of MPO in plasma Parameter

SM

ELISA

Linear range of MPO concentrations, ng/ml Threshold sensitivity, ng/ml Measurement differences in one experiment, % Measurement differences in different experiments, % Analysis time/number of samples

25–700 25 3 4 10–15 min/6 samples

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the substrate, but the contributions of other agents with pseudoperoxidating activity were left beyond consideration. Our studies indicate that this approach is erroneous, because the actual MPO activity in plasma is not properly determined. The analysis of MPO activity in blood plasma appears promising in the development of methods for assaying the enzyme under physiological conditions, in the case of various diseases, and during clinical trials of pharmaceuticals. Although the spectrophotometrical method requires no special equipment, antibodies, or MPO standards, it is reasonable to determine both the active and immunoreactive MPO concentrations. Their combination would provide information not only on the MPO level and activity, but also on the operation of systems regulating and inhibiting MPO in plasma; i.e., it is one of the indices of the antioxidant state. EXPERIMENTAL Reagents: o-Dianisidine, 4-aminobenzoic acid hydrazide, salicylhydroxamic acid, tyrosine, human hemoglobin, o-phenylenediamine, and Freund’s adjuvant were purchased from Sigma-Aldrich (United States); 4-chloro-1-naphthol from Fluka (Switzerland); guaiacol from Merck (Germany); chromatographic sor-

bents from Pharmacia (Sweden); 3% hydrogen peroxide from Sagmel (United States); horseradish peroxidase-labeled goat antibodies against rabbit IgG antibodies and nonfat dry milk from BioRad (United States); and reagents for electrophoresis from Medigen (Russia). Isolation of MPO from leukocytes was performed by chromatography on Heparin-Sepharose and PhenylSepharose and by gel filtration [26]. The purity was characterized by the A430/A280 ratio, Rz, which was generally no less than 0.75. The Rz of our MPO preparation was 0.85. Plasma samples were obtained by centrifuging blood stabilized with 3.8% sodium citrate at 600 g for 15 min. Measurement of the peroxidase activity of native MPO. Standard samples for the spectrophotometrical analysis of MPO peroxidase activity contained a 50-mM sodium phosphate buffer, pH 7.0, 1 mM ç2é2, a substrate, and MPO at the required concentration. The following substrates were used: 500 µM guaiacol, 500 µM 4-chloro-1-naphthol, or 380 µM DA. The reaction was initiated by adding 1 mM H2O2. Absorbance values A450, A600, and A460 were measured at 20°C in the kinetic regime for guaiacol, 4-chloro-1-naphthol, and DA, respectively, with a SOLAR PV 1251c spectropho-

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Fig. 8. (a, b) Correlation between the MPO concentration in plasma measured by ELISA and MPO peroxidase activity determined by the difference spectrophotometrical method with (a) 100 µM salicylhydroxamic acid (n = 38, r = 0.694, P < 0.0001) and (b) 50 µM 4-aminobenzoic acid hydrazide (n = 38, r = 0.701, P < 0.0001). (c) Correlation between the peroxidase MPO activities determined by the difference spectrophotometrical method with 100 µM salicylhydroxamic acid and with 50 µM 4-aminobenzoic acid hydrazide (n = 38, r = 0.903, P < 0.0001).

tometer (Belarus). Fluorescence changes of MPO-catalyzed oxidation of 100 µM tyrosine were recorded using an LSF 1211A spectrofluorometer (SOLAR, Belarus) with excitation at 325 nm and emission at 410 nm. The measurement conditions were the same. Measurement of plasma peroxidase activity. The peroxidase activities of MPO and hemoglobin in plasma were measured in a 50 mM sodium phosphate buffer, pH 7.0. Peroxidase activity measurement at pH 4.5 and the study of the pH dependence of MPO and hemoglobin activities in the pH range of 4.0–7.0 were carried out in a buffer solution (hereafter referred to as phosphate citrate buffer) containing 0.2 M Na2HPO4 + 0.1 M citric acid. The reaction was initiated by the addition of 100 µM ç2é2 unless otherwise specified. Original plasma samples were diluted by a factor of 13.3, that is, a 60 µl volume of plasma was adjusted to 800 µl. MPO activity was inhibited by adding 100 µM salicylhydroxamic acid or 50 µM 4-aminobenzoic acid hydrazide. The MPO activity in plasma was calculated by the equation ÄMPO = (∆A/min – ∆Ainh/min) × (V/v), where ∆A/min and ∆Ainh/min are the rates of DA oxidation without and with the inhibitor; V is the total reaction volume; and v, the plasma sample volume. The rate of DA oxidation was determined by linear extrapolation as the slope of the starting linear region of the kinetic curve with at least six experimental points. For this purpose, the statistical unit of the Origin 7.0 software was used. Antigen for animal immunization. MPO was additionally purified from minor admixtures by preparative PAGE. The gel was prepared as follows: resolving gel, 7.5% polyacrylamide, KOH-CH3COOH buffer, pH RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY

4.3; stacking gel, 5% polyacrylamide, KOHCH3COOH buffer, pH 6.7 [26]. According to our experience, a gradual release of the antigen from the gel in the immunization site and the presence of polyacrylamide in the sample increase the antibody titer. The green zone of the gel, containing MPO, was cut and frozen in portions containing 200 µg of protein for one immunization. Prior to immunization, gel fragments with the antigen were homogenized with phosphatebuffered saline (PBS; 150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.4) and Freund’s adjuvant 1 : 10 v/v. Rabbit IgG antibodies against MPO. Rabbits were immunized by paraspinal subcutaneous injections of the antigen-containing suspension at 14-day intervals. The first immunization was done with Freund’s complete adjuvant, and the second and third, with incomplete. Fifty milliliters of blood were taken from the auricular vein 10–12 days after the third immunization. The serum was separated and IgG antibodies were precipitated by adding (NH4)2SO4 to a concentration of 1.75 M. The pellet was washed three times with 1.75 M (NH4)2SO4, dissolved in PBS, and dialyzed against a 17.5 M sodium phosphate buffer, pH 6.3. The solution was loaded onto a column with DEAE-Sephadex A-50. The void volume contained IgG antibodies. An additional amount of IgG antibodies was obtained by elution with a 35 mM sodium phosphate buffer, pH 7.6. The fractions contained IgG antibodies with M = 160 kDa according to SDS-PAGE [37]. They were combined, dialyzed against water, and lyophilized. Rat IgG antibodies against MPO. One-year-old rats weighing 500–600 g were immunized by paraspinal subcutaneous injections of the antigen-containing suspension at 10-day intervals. The first immunization

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was done with Freund’s complete adjuvant, and the next three, with incomplete. Rats were narcotized with diethyl ether 10–12 days after the fourth immunization, 25 ml of blood was collected, and the serum was separated. To prepare the affinity sorbent, MPO was immobilized on BrCN-Sepharose in the ratio of 5 mg per 1 ml of the resin [26]. A 5 × 2-cm column with MPOSepharose was equilibrated with PBS, loaded with 150 ml of the serum, and washed with 1 M NaCl in PBS. The elution was performed with 0.2 M glycineHCl, pH 2.4. Fractions 2.5 ml in volume were collected into tubes with 2.5 ml of 0.4 M Tris-HCl, pH 8.0. Fractions containing IgG antibodies with Mr = 160 kDa according to SDS-PAGE were combined, dialyzed against a 10 mM sodium phosphate buffer, pH 7.4, and stored at –70°ë. ELISA protocol. Analysis was carried out in 96well flat-bottom polystyrene plates. Reagents were added with multichannel dispensers. The plates were incubated in a Thermo Shaker (BioSan, Latvia) at 37°ë and 290 rpm. The solutions were prepared with BLOTTO-T: 3% w/v nonfat dry milk in PBS supplemented with 0.05% Tween 20. The operations are listed in Table 1. The sorption of rat IgG antibodies in wells was usually carried out at 4°ë overnight. In some cases, plates with IgG antibodies were stored at 4°ë for up to 14 days without a loss in the analysis accuracy. After the termination of the chromogenic reaction, Ä492 was measured in wells with a StatFax microplate reader (United States). The dependence of A492 on lg[MPO] was linearly approximated by the least squares method implemented in Microsoft Excel 2002. The coefficient of determination R2 was no less than 0.99. The contents of MPO in samples were calculated from the resulting equation A492 = k × log [ MPO] + b. Statistical evaluation of the results was performed by Student’s and Pearson’s tests and correlation analysis. ACKNOWLEDGMENTS This work was supported by the Russian Foundation for Basic Research, project no. 08-04-00532, and the Belarusian Foundation for Basic Research, project no. B08-107. We are grateful to Prof. V.N. Kokryakov, Institute for Experimental Medicine, St. Petersburg, Russia, who kindly provided us with white-cell-rich suspension for MPO isolation. REFERENCES 1. Andrews, P.C. and Krinsky, N.I., J. Biol. Chem., 1981, vol. 256, pp. 4211–4218. 2. Schultz, J. and Kaminker, K., Arch. Biochem. Biophys., 1962, vol. 96, pp. 465–467. 3. Deby-Dupont, G., Deby, C., and Lamy, M., Intensivmed., 1999, vol. 36, pp. 500–513.

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