1980
Chem. Res. Toxicol. 2007, 20, 1980–1988
Kinetics of Hypobromous Acid-Mediated Oxidation of Lipid Components and Antioxidants Ojia Skaff, David I. Pattison,* and Michael J. Davies The Heart Research Institute, 114 Pyrmont Bridge Road, Camperdown, Sydney, NSW 2050, Australia ReceiVed August 29, 2007
Hypohalous acids are generated from the oxidation of halide ions by myeloperoxidase and eosinophil peroxidase in the presence of H2O2. These oxidants are potent antibacterial agents, but excessive production can result in host tissue damage, with this implicated in a number of human pathologies. Rate constants for HOCl with lipid components and antioxidants have been established. Here, the corresponding reactions of HOBr have been examined to determine whether this species shows similar reactivity. The secondorder rate constants for the reaction of HOBr with 3-pentenoic acid and sorbate, models of unsaturated lipids, are 1.1 × 104 and 1.3 × 103 M-1 s-1, respectively, while those for reaction of HOBr with phosphoryl-serine and phosphoryl-ethanolamine are ca. 106 M-1 s-1. The second-order rate constants (M-1 s-1) for reactions of HOBr with Trolox (6.4 × 104), hydroquinone (2.4 × 105), and ubiquinol-0 (2.5 × 106) were determined, as models of the lipid-soluble antioxidants, R-tocopherol, and ubiquinol10; all of these rate constants are ca. 50-2000-fold greater than for HOCl. In contrast, the second-order rate constants for the reaction of HOBr with the water-soluble antioxidants, ascorbate and urate, are ca. 106 M-1 s-1 and closer in magnitude to those for HOCl. Kinetic models have been developed to predict the sites of HOBr attack on low-density lipoproteins. The data obtained indicate that HOBr reacts to a much greater extent with fatty acid side chains and lipid-soluble antioxidants than HOCl; this has important implications for HOBr-mediated damage to cells and lipoproteins. Introduction 1
The heme enzyme myeloperoxidase (MPO) is released from the intracellular granules of neutrophils, monocytes, and some macrophages upon activation. MPO catalyzes the oxidation of halide and pseudohalide ions, mainly chloride (Cl-), bromide (Br-), and thiocyanate (SCN-), by hydrogen peroxide to yield the potent oxidants hypochlorous (HOCl), hypobromous (HOBr), and hypothiocyanous acid (HOSCN) (1–4). HOCl and HOBr react readily with biological materials such as amino acids, proteins, carbohydrates, lipids, and DNA (5–12). At physiological pH values, these species are present as equilibrium mixtures of HOCl/-OCl and HOBr/-OBr (11, 13); henceforth, the terms HOCl and HOBr are used to describe the mixture of these species. HOCl and HOBr are potent antibacterial agents crucial for the immune response (1). However, excessive or misplaced generation can lead to tissue damage, and this has been implicated in several human inflammatory diseases including atherosclerosis (14–16), arthritis (17), cystic fibrosis (18), asthma (19–21), and some cancers (22–24). In vitro studies have shown that at pH 7.8, 40% of the hydrogen peroxide consumed by isolated MPO is converted to HOBr (25); subsequent extension of these studies to isolated neutrophils has confirmed that HOBr is a significant product of these cells when physiological concentrations of Cl- and Br- are present (Chapman, A.L.P., Skaff, O., Senthilmohan, R., Kettle, A. J., and Davies, M. J. * To whom correspondence should be addressed. Tel: +61-2-8208-8900. Fax: +61-2-9565-5584. E-mail:
[email protected]. 1 Abbreviations: apoB100, apolipoprotein B-100; HOBr, the physiological mixture of hypobromous acid and its anion; HOCl, the physiological mixture of hypochlorous acid and its anion; HOX, hypohalous acid; HQ, hydroquinone; LDL, low-density lipoprotein; MPO, myeloperoxidase; PA, 3-pentenoic acid; p-EA, phosphoryl-ethanolamine; p-Ser, phosphoryl-serine.
Submitted for publication). These data indicate that HOBr, and species derived from it, may contribute in a significant manner to bacterial killing and inflammatory tissue damage. While these studies demonstrate that HOBr is generated by MPO and neutrophils, it should be noted that the formation of HOSCN has been proposed to account for up to 50% of the consumed H2O2 by MPO in the presence of physiological (pseudo)halide concentrations at pH 7.4 (26). Furthermore, it has been suggested that SCN- is an efficient endogenous scavenger of HOBr and may limit its capacity to inflict host tissue damage (27). The biological events that lead to tissue damage induced by HOCl and HOBr, such as in atherosclerosis, are poorly characterized. Oxidation of low-density lipoproteins (LDL) is known to result in an increased uptake of the modified particles by macrophages to yield lipid-laden foam cells, which are an early hallmark and a defining feature of atherosclerosis (28–30). Although the oxidants responsible for LDL modification in vivo have not been substantiated, there is increasing evidence for a role for MPO-derived species (14–16, 31, 32). Thus, people with established atherosclerosis have elevated levels of circulating MPO in their blood, and MPO is now recognized as a significant, independent risk factor for coronary heart disease (14). Both MPO (32) and halogenated tyrosine residues (16), an established marker of hypohalous acid (HOX)-mediated protein oxidation, can be readily detected in atherosclerotic lesions. To fully elucidate the role of HOX in disease, a detailed knowledge of the kinetics and products of these reactions is required. Second-order rate constants for the reaction of HOCl and HOBr with protein components have been reported (5, 12, 33, 34). For HOCl, these decrease in the order Met > Cys >> cystine ∼ His ∼ R-amino > Trp > Lys >> Tyr ∼ Arg > backbone amides > Gln ∼ Asn at pH 7.4 (5). In contrast,
10.1021/tx7003097 CCC: $37.00 2007 American Chemical Society Published on Web 11/30/2007
ReactiVity of HOBr with Lipids and Antioxidants
HOBr reacts, in general, 30–100-fold faster than HOCl, but the extent of the increase is markedly substrate-dependent (33). Thus, Cys and Met residues are ca. 10-fold less reactive with HOBr than with HOCl, while ring halogenation of Tyr is ca. 5000-fold faster with HOBr. Previous studies suggest that some of the cellular effects of HOCl and HOBr arise from modification of polyunsaturated fatty acids (35–41), either directly or via the intermediacy of chloramines/bromamines (42). At physiological pH, HOCl reacts with both the amine functions of the headgroups of phosphatidyl serine and ethanolamine to form chloramines [HOCl + RNH2 f RNHCl + H2O] or the double bonds of unsaturated fatty acids to form chlorohydrins [HOCl + RCHdCHR′ f RCH(OH)CH(Cl)R′]. Second-order rate constants for the reaction of HOCl with these components have been determined, with those for the nitrogen-containing headgroups of lipids similar to those determined for protein amines (k, ca. 105 M-1 s-1). In contrast, addition across double bonds is orders of magnitude slower (6). Whether HOBr follows a similar pattern of reactivity is unclear; the detection of both bromamines and bromohydrins on LDL and isolated phospholipids after exposure to HOBr suggests that this may not be true (35, 38). HOCl reacts rapidly with the water-soluble antioxidants, ascorbate and urate (k, 105-107 M-1 s-1), indicating that these species may modulate HOCl-mediated damage (7, 8, 12). In contrast, reaction with lipid-soluble antioxidants such as R-tocopherol (a component of vitamin E) and ubiquinol-10 (reduced coenzyme Q10) appears, on the basis of data obtained for the model compounds Trolox, hydroquinone (HQ), and ubiquinol0, to be much slower (k, ca. 103 M-1 s-1). It has been suggested, and experimentally confirmed (43), that these reactions are uncompetitive when compared to protein damage to LDL (6). Rate constants for the analogous reactions of HOBr have not been reported. The current study reports second-order rate constants for the reaction of HOBr with both lipid components and antioxidants and compares these data with those for HOCl; these values differ markedly. These kinetic data have been incorporated into computational models for the reaction of HOBr with LDL, with the resulting data indicating substantial differences between HOCl- and HOBr-mediated damage. These findings provide insight into HOBr-mediated damage to complex biological targets, such as membranes.
Experimental Procedures Materials. All chemicals were obtained from Sigma/Aldrich/ Fluka, with the exception of NaBr (>99% purity; from Merck), and were used without further purification. HOCl stock solutions (0.6 M in 0.1 M NaOH, low in bromide) were standardized by measuring the absorbance at 292 nm at pH 12 [292 (-OCl) ) 350 M-1 cm-1] (13). All kinetic studies were performed in 0.1 M phosphate buffer (pH 7.4), except the competitive HPLC studies, which were carried out in 10 mM phosphate buffer (pH 7.4). All phosphate buffers were prepared using Milli Q water and treated with Chelex resin (Bio-Rad) to remove contaminating trace metal ions. HOBr Preparation. Solutions of HOBr were prepared by mixing HOCl (80 mM in H2O, pH 13) with a small excess of NaBr (90 mM in H2O) in equal volumes. The reaction was left for 1 min before dilution with 0.1 M phosphate buffer (pH 7.4) to the desired concentration of HOBr (typically 0.2–2.0 mM). As HOBr disproportionates slowly to form Br- and BrO2- (44–46), fresh solutions were prepared for each kinetic run and used within 30 min. At pH 7.4, hypobromous acid exists primarily as HOBr with low concentrations of -OBr also present (pKa 8.7) (11). Stopped-Flow Studies. Stopped-flow studies were carried out using either a fully upgraded Applied Photophysics SX.18MV
Chem. Res. Toxicol., Vol. 20, No. 12, 2007 1981 system (0.01–10 s) or a Hi-Tech SFA 20 attachment to a PC-controlled Perkin-Elmer Lambda 40 UV/vis spectrometer (5–900 s) as described previously (6). The recent upgrade to the SX.18MV system renders it almost equivalent in performance to an Applied Photophysics SX20 stopped-flow machine. For the kinetic experiments (except in the case of sorbate and ubiquinol-0), HOBr was kept as the limiting reagent, with at least a 2-fold excess of substrate (typically 0.02–1 mM HOBr and 0.05–25 mM substrate). This limits secondary reactions [e.g., dibromamine formation (47)] and, with excesses of >5-fold, reduces the kinetics to pseudo first-order, facilitating analysis. With ubiquinol-0 and sorbate, these were used as the limiting reagents (ca. 5 µM for ubiquinol-0 and 25 µM for sorbate), with excesses of HOBr from 2- to 25-fold. This reduced the kinetics to pseudo first-order but assumed that the initial products were inert to further oxidation. Data were analyzed by global methods (Specfit32, version 3.0.37, Spectrum Software Associates) or single wavelength analysis (OriginPro 7.0). All second-order rate constants reported are averages of at least four separate determinations, and errors are specified as 95% confidence limits. Experiments with Ubiquinol-0. Solutions of ubiquinone-0 (45 µM) and HOBr were deoxygenated by continuous bubbling with N2 for approximately 15 min. NaBH4 (0.1 mg mL-1) was then added to generate ubiquinol-0. The concentration of NaBH4 chosen ensured that full reduction of ubiquinone-0 did not occur and that NaBH4 was fully consumed. The resulting solutions were used as quickly as possible to minimize any reoxygenation. The concentrations of ubiquinone-0 and ubiquinol-0 were established from the UV spectrum of the reduced solution, using the extinction coefficients determined for each compound at 268 and 288 nm (6). The reaction of ubiquinol-0 (ca. 5 µM) with HOBr (50–125 µM) was examined over 25–100 ms between 240 and 320 nm (10 nm intervals). Stock solutions of ubiquinol-0 were checked for autoxidation at the completion of the experiment; minimal degradation was observed under the conditions employed. HPLC Instrumentation and Methods. N-R-Acetyl-Tyr, N-Racetyl-3-bromotyrosine (N-Ac-Br-Tyr), and N-R-acetyl-3,5-dibromotyrosine (N-Ac-Br2-Tyr) were quantified using a gradient HPLC method as described previously (33). Using the conditions employed, N-R-acetyl-Tyr eluted at 13.0 min, N-R-acetyl-Br-Tyr eluted at 18.1 min, and N-R-acetyl-Br2-Tyr eluted at 28.5 min. Computational Modeling of HOBr Reactivity with LDL. Computational modeling studies were performed with Specfit software. The LDL composition was that used previously for HOCl (6). Rate constants for the reaction of protein components were taken from ref 33. The software was allowed to undergo 10000 iterations to yield predicted reactant and product concentrations over time intervals chosen to ensure complete consumption of HOBr.
Results Kinetics of HOBr Reactions with Phospholipid Headgroups. Second-order rate constants for the reaction of HOBr with phosphoryl-ethanolamine (p-EA) and phosphoryl-serine (pSer), model compounds for lipid headgroups, were determined at 10 and 22 °C ([HOBr] ) 0.1 mM and [RNH2] ) 0.2–0.8 mM). For both compounds, a small loss in absorbance was detected in the region 280–300 nm, with a concomitant increase in absorbance between 240 and 270 nm. This is consistent with the rapid (within 200 ms) conversion of -OBr to the corresponding bromamine. The data for both compounds were fitted well by global analysis with a simple mechanism (HOBr + amine f bromamine) to give the second-order rate constants reported in Table 1. These results were confirmed by fitting the data obtained at 250 nm for p-EA, and at 240 nm for p-Ser, to a single exponential function. The resulting observed rate constants (kobs) were plotted against the amine concentration (Figure 1), with the gradients of the resulting straight lines yielding the second-order rate constants; these agree well with
1982 Chem. Res. Toxicol., Vol. 20, No. 12, 2007
Skaff et al.
Table 1. Second-Order Rate Constants (with 95% Confidence Limits) at 22 °C and pH 7.4 (in 0.1 M Phosphate Buffer) for the Reactions of HOBr with Lipid Components and Antioxidants
Figure 1. Plots of the observed rate constant (kobs) vs substrate concentrations for (a) p-EA and (b) p-Ser at 10 (triangles) and 22 °C (circles). kobs was determined by fitting the absorbance changes at 250 nm for p-EA and 240 nm for p-Ser to a single exponential growth. Error bars represent standard deviations from repeated experiments. Some errors in fits are smaller than the size of the symbol.
The rate constants were determined for p-EA at 10 °C by global analysis, k ) (2.6 ( 0.4) × 105 M-1 s-1, and single wavelength analysis, k ) (2.3 ( 0.1) × 105 M-1 s-1. b Determined by global analysis. c Determined by single wavelength analysis. d The rate constants were determined for p-Ser at 10 °C by global analysis, k ) (3.1 ( 0.4) × 105 M-1 s–1, and single wavelength analysis, k ) (2.8 ( 0.2) × 105 M-1 s-1. e Determined from UV data by competition kinetics. a
those obtained by global analysis (Table 1). At 10 °C, the second-order rate constants were ca. 3 times slower than those measured at 22 °C (Table 1). Kinetics of HOBr Reactions with Double Bonds. 3-Pentenoic acid (PA) and sorbate (see Table 1) were used as models for the double bonds of unsaturated fatty acids. The reaction of HOBr with PA was investigated at 22 °C ([HOBr] ) 1.0 mM and [PA] ) 2.5–25.0 mM). The -OBr absorbance at 290 nm decayed over a period of 0.1–0.5 s depending on the substrate concentration, but no new peaks due to bromohydrin formation were observed. The data were readily fitted with pseudo firstorder kinetics (HOBr + RCH)CHR′ f bromohydrin) to yield the second-order rate constant (Table 1). Second-order rate constants determined from the slopes of linear pseudo firstorder plots of kobs against [PA], k2 ) kobs/[PA], were consistent with the global analysis data (Table 1). The reaction of HOBr with the conjugated diene group of sorbate was investigated at 22 °C by keeping sorbate as the limiting reagent, as it has a strong absorbance at 240–260 nm ([HOBr] ) 50–500 µM and [sorbate] ) 25 µM). Small absorbance changes were detected over 20–240 s due to the consumption of sorbate, but no new spectral peaks were
observed. The data were fitted as described above and yielded the second-order rate constants given in Table 1; these are an order of magnitude less than for PA. Kinetics of HOBr Reactions with Lipid-Soluble Antioxidants. R-Tocopherol and ubiquinol-10 are the major lipidsoluble antioxidants present in LDL and cell membranes (48). Reaction with these materials was investigated using watersoluble analogues. The reaction of HOBr with Trolox, a watersoluble model of R-tocopherol, was investigated at low concentrations ([HOBr] ) 50 µM and [Trolox] ) 100–250 µM), since both Trolox and its oxidized product absorb strongly in the UV region. Stopped-flow studies (22 °C, λ ) 240–320 nm) yielded complex kinetics over 20 s (Figure 2a). A rapid growth (99% of HOCl consumption) at Met and Cys residues of the apoB100 protein at low molar excesses of HOCl (
