Biochemistry 2004, 43, 4799-4809
4799
Kinetic Analysis of the Reactions of Hypobromous Acid with Protein Components: Implications for Cellular Damage and Use of 3-Bromotyrosine as a Marker of Oxidative Stress† David I. Pattison* and Michael J. Davies The Heart Research Institute, 145 Missenden Road, Camperdown, Sydney, NSW 2050, Australia ReceiVed October 30, 2003; ReVised Manuscript ReceiVed January 21, 2004
ABSTRACT: Hypohalous acids (HOX, X ) Cl, Br) are produced by activated neutrophils, monocytes, eosinophils, and possibly macrophages. These oxidants react readily with biological molecules, with amino acids and proteins being major targets. Elevated levels of halogenated Tyr residues have been detected in proteins isolated from patients with atherosclerosis, asthma, and cystic fibrosis, implicating the production of HOX in these diseases. The quantitatiVe significance of these findings requires knowledge of the kinetics of reaction of HOX with protein targets, and such data have not been previously available for HOBr. In this study, rate constants for reaction of HOBr with protein components have been determined. The secondorder rate constants (22 °C, pH 7.4) for reaction with protein sites vary by 8 orders of magnitude and decrease in the order Cys > Trp ∼ Met ∼ His ∼ R-amino > disulfide > Lys ∼ Tyr . Arg > backbone amides > Gln/Asn. For most residues HOBr reacts 30-100 fold faster than HOCl, though Cys and Met residues are ∼10-fold less reactive, and ring halogenation of Tyr is ∼5000-fold faster. Thus, Tyr residues are more, and Cys and Met much less, important targets for HOBr than HOCl. Kinetic models have been developed to predict the targets of HOX attack on proteins and free amino acids. Overall, these results shed light on the mechanisms of cell damage induced by HOX and indicate, for example, that the 3-chloroTyr:3-bromo-Tyr ratio does not reflect the relative roles of HOCl and HOBr in disease processes.
Hypohalous acids (HOX, X ) Cl, Br) are strong oxidants with potent antibacterial properties (1). HOCl and HOBr are integral factors in mammalian host defense systems and are produced in vivo by activated neutrophils, monocytes, and possibly macrophages (HOCl) and activated eosinophils (HOBr) (2, 3). HOCl and HOBr are formed by the reactions of H2O2 with Cl- or Br- catalyzed by the heme enzymes, myeloperoxidase (MPO)1 (4) and eosinophil peroxidase (EPO) (5). Both HOCl and HOBr react readily with biological molecules including amino acids, proteins, antioxidants (including thiols), carbohydrates, lipids, and DNA (6-14). Although HOX formation is crucial for immune response, excessive or misplaced generation can damage host tissues, and this has been implicated in several human inflammatory diseases, including arthritis (15), some cancers (16-18), heart disease (19-21), cystic fibrosis (22), and asthma (23-25). † This work was funded by Australian Research Council Grants A00001441 and F00001444. * To whom correspondence should be addressed. Telephone: +612-9550-3560. Fax: +61-2-9550-3302. E-mail:
[email protected]. 1 Abbreviations: apo-A1, apolipoprotein-A1; Br-HPPA, 3-(3-bromo4-hydroxyphenyl)propionic acid; Br2-HPPA, 3-(3,5-dibromo-4-hydroxyphenyl)propionic acid; Br-Tyr, 3-bromotyrosine; BSA, bovine serum albumin; CANH2, -amino-n-caproic acid; Cl-Tyr, 3-chlorotyrosine; DTDPA, 3,3′-dithiodipropionic acid; EPO, eosinophil peroxidase; ESIMS, electrospray ionization mass spectrometry; GSSG, oxidized glutathione; HPLC, high-performance liquid chromatography; HPPA, 3-(4-hydroxyphenyl)propionic acid; HSA, human serum albumin; IAA, 4-imidazoleacetic acid; LDL, low-density lipoprotein; MPO, myeloperoxidase; N-acetyl-Br-Tyr, N-acetyl-3-bromotyrosine; N-acetyl-Br2Tyr, N-acetyl-3,5-dibromotyrosine.
Elevated levels of EPO and brominated Tyr (Br-Tyr) residues have been detected in the sputum of asthmatic patients relative to controls (23-25), especially when the patients were subject to an allergen challenge (24, 25). These observations are consistent with a role for EPO and HOBr in asthma. In light of the potential role of HOX in these diseases, it is important to have a thorough understanding of the chemical modifications induced by these oxidants in biological tissues and, particularly, the reactions that give rise to compounds, such as halogenated Tyr residues, that are used as markers for HOX-mediated damage. To achieve this, a detailed knowledge of the kinetics and products of these reactions is essential. The products of the reactions of HOCl with Cys and Met are disulfides and oxy acids, and sulfoxides, respectively (9, 26, 27). In the presence of amines it is also possible to generate sulfonamides, via reaction of the initial sulfenyl chloride intermediates with neighboring amine groups (28-30). Similar reactions are believed to occur with HOBr. The reaction of HOCl with amines (e.g., Lys and R-amino groups) generates unstable chloramine species (RNHCl, RR′NCl) and, when HOCl is in excess, dichloramines (RNCl2) (31-33). Chloramines on R-amino groups typically decompose within a few minutes of formation to yield aldehydes (34), but others such as those on the -amine group of Lys are more stable, with t1/2 >1 h (35). HOCl also reacts with amide groups (e.g., protein amide groups) to yield chloramides (6, 14). Analogous processes occur with HOBr (5, 12, 13), yielding bromamines/amides that are
10.1021/bi035946a CCC: $27.50 © 2004 American Chemical Society Published on Web 03/26/2004
4800 Biochemistry, Vol. 43, No. 16, 2004 typically more reactive than chloramines/amides (14, 36). The reactions of HOX with Tyr residues generate stable halogenated Tyr residues that are widely used as markers for HOX-mediated damage (21, 22, 24, 37-46). The major products are 3-chloro- and 3-bromotyrosine (Cl-Tyr and BrTyr), but dihalogenated compounds (3,5-dichloro- and 3,5dibromotyrosine) are formed with high excesses of HOX. The stable products of reaction of HOX with cystine and other amino acid side chains (e.g., His, Trp, and Arg) are poorly characterized for both HOCl and HOBr but probably include 2-oxindole derivatives of Trp and 2-oxohistidine (33). The reaction kinetics for HOCl with biological materials are well established (6, 8-10, 32, 47-49), with the secondorder rate constants at pH 7.4 decreasing in the order Met > Cys . cystine ∼ His ∼ R-amino > Trp > Lys . Tyr ∼ Arg > backbone amides > Gln ∼ Asn (6). Incorporation of these data into a computational kinetic model has allowed the initial sites of attack of varying molar excesses of HOCl on human serum albumin (HSA), apolipoprotein-A1 (apoA1), and free amino acids to be predicted; these data correlate well with experimental observations (6). Rate constants have also been determined for reaction of HOCl with lipid components and some antioxidants, and these data have been used to model the reactivity of HOCl with lipoproteins and the nucleosome (7, 50). In contrast to HOCl, there is a paucity of kinetic data for HOBr. It has been reported that the rates of reaction of HOBr with cyclic dipeptides and NADH are ca. 10 times faster than with HOCl (12-14). Prutz has provided evidence that bromination of the Tyr side chain with HOBr is also more rapid (ca. 250 times) than chlorination by HOCl (36) and has intimated that reaction of HOBr with Met side chains is slower than with HOCl (12). In the current work, second-order rate constants have been determined for the reactions of HOBr with model protein substrates at pH 7.4. These data have been incorporated into a computational model to predict the reactivity of HOBr with the plasma protein, HSA, and with plasma concentrations of free amino acids. These predictions have been compared with experimental data and the rate constants compared with those for HOCl. Overall, these show that Br-Tyr is a much more sensitive marker for bromination than Cl-Tyr is for chlorination. This may result in the extent of HOBr-mediated oxidation being overestimated (or HOCl-mediated processes underestimated) on the basis of the yields of Cl-Tyr and BrTyr. Furthermore, these data suggest that the more rapid (∼10-fold faster) rate of erythrocyte lysis induced by HOBr compared to HOCl (51, 52) is not due to oxidation or depletion of Cys and Met residues (either low molecular weight or on proteins) and that damage to His, Lys, Tyr, Trp, or cystine residues on proteins could be the key determinant of the rate of cell lysis. EXPERIMENTAL PROCEDURES Materials. All chemicals were obtained from Sigma/ Aldrich/Fluka and used as received, with the exception of N-acetyl-Ala-OMe and cyclo(Asp)2 (Bachem), sodium bromide (>99% purity; Merck), and sodium hypochlorite (in 0.1 M NaOH, low in bromine; BDH Chemicals). The HOCl was standardized by measuring the absorbance at 292 nm at pH 12 [292(-OCl) ) 350 M-1 cm-1] (53). All studies were
Pattison and Davies performed in 100 mM phosphate buffer (pH 7.4), except for the 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 transition metal ions. The pH values of solutions were adjusted, where necessary, to pH 7.4 using 100 mM H2SO4 or 100 mM NaOH. HOBr Preparation. HOBr was prepared by mixing HOCl (40 mM in H2O, pH 13) with NaBr (45 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 required concentration of HOBr (typically 0.2-2.0 mM). As HOBr disproportionates slowly to form Br- and BrO2- (14, 36, 54), fresh solutions were prepared for each kinetic run and used within 30 min. To investigate whether Br2 formed in the presence of excess Br- (55) contributed to the observed reaction kinetics, HOBr solutions were also prepared with increasing concentrations of NaBr (45-250 mM). At neutral pH, hypobromous acid exists primarily as HOBr with low concentrations of -OBr also present (pKa 8.7) (12). Stopped-Flow Studies. Two stopped-flow systems (both 10 mm path length) were used depending on the time scale being studied (6). An Applied Photophysics SX.18MV system was used for kinetics on the 10 ms-10 s time scale and a Hi-Tech SFA 20 attachment connected to a PCcontrolled Perkin-Elmer Lambda 40 UV/vis spectrometer for reactions on the 5-900 s time scale. For both systems, the sample chamber was maintained at the requisite temperature by circulating water from a thermostated water bath. Kinetic data were accumulated at 10 nm intervals between 220 and 350 nm (0.1 M phosphate buffer baseline) and combined to give time-dependent spectral data. For the majority of the kinetic experiments (except where stated), HOBr was kept as the limiting reagent, with at least a 2-fold excess of substrate (typically 0.1-1.0 mM HOBr, 0.2-10.0 mM substrate). This reduces the kinetics to pseudo first order and limits the occurrence of secondary reactions (e.g., dibromination). Data were processed by global analysis methods, using Specfit software (version 3.0.15; Spectrum Software Associates; see http://www. bio-logic.fr/rapid-kinetics/specfit.html for further details). All second-order rate constants reported are averages of at least four determinations, and errors are specified as 95% confidence limits. UV/Vis Spectroscopy. UV/vis spectroscopy was undertaken on a Perkin-Elmer Lambda 40 spectrometer. Spectra were typically acquired (relative to a 0.1 M phosphate buffer baseline) between 220 and 350 nm (1-2 nm intervals), with a time interval of 1-30 min depending on the reaction time scale. The cuvettes were thermostated to 22 °C using a Peltier block. Data were imported into Specfit software and the kinetics processed by global analysis techniques (as above). HPLC Instrumentation and Methods. Analysis and quantification of N-acetyl-Tyr and its reaction products with HOBr were carried out on a Shimadzu LC-10A HPLC system (Shimadzu, South Rydalmere, NSW, Australia). The reaction mixtures were separated on a Zorbax reverse-phase HPLC column (25 cm × 4.6 mm, 5 µM particle size; Rockland Technologies, Newport, DE) packed with octadecyl silanized silica, equipped with a Pelliguard guard column (2 cm; Supelco). The column was maintained at 30 °C using a column oven (Waters Corp., Milford, MA). The mobile phase
Second-Order Rate Constants for HOBr with Proteins was comprised of a gradient of solvent A (10 mM phosphoric acid with 100 mM sodium perchlorate at pH 2.0) and solvent B [80% (v/v) methanol in nanopure H2O] eluting at 1 mL min-1. The gradient was programmed as follows: 0-5 min, elution with 90% solvent A and 10% solvent B; over the next 5 min the proportion of solvent B was increased to 35%; isocratic elution at 35% solvent B occurred for 35 min; over the following 1 min, solvent B was increased to 50%, and the column was washed at 50% solvent B for 9 min; over the subsequent 5 min the proportion of solvent B was reduced to 10%, and the column was allowed to reequilibrate for 5 min prior to injection of the next sample. The eluent was monitored in series by a UV detector (280 nm), a fluorescence detector (λex, 280 nm; λem, 320 nm), and a dual-channel electrochemical detector (Coulochem II; ESA, Chelmsford, MA). The first channel of the electrochemical detector was set to an oxidation potential of +550 mV (sensitivity, 2 µA) to oxidize any coeluting peaks, and the second channel was set to an oxidation potential of +700 mV (sensitivity, 5 µA) to quantify the halogenated N-acetyl-Tyr products. Peak areas were determined using Class VP 6.0 software (Shimadzu) and compared to authentic standards when required. Using these conditions, N-acetyl-Tyr was detected in the +700 mV electrochemical channel at a retention time of 14.6 min, N-acetyl-3-bromotyrosine (N-acetyl-Br-Tyr) at 21.4 min, and N-acetyl-3,5-dibromotyrosine (N-acetyl-Br2-Tyr) at 38.0 min. A small impurity peak present in the parent compound was also detected with a retention time of 8.9 min, but this was not characterized further. The product peaks assigned to N-acetyl-Br-Tyr and N-acetyl-Br2-Tyr were characterized by electrospray ionization mass spectrometry (ESI-MS). Approximately 1 mL fractions from eight injections (50 µL) were collected and pooled together. Phosphate and perchlorate salts were removed by reducing the volume of the collected fractions to ca. 65% (to remove MeOH), then loading onto Sep-Pak C18 cartridges (Waters Corp., Milford, MA) that had been preconditioned with MeOH, and equilibrated with solvent A from the HPLC gradient. The cartridges were subsequently washed with nanopure H2O to elute the water-soluble salts, and the product peaks were then eluted using 100% MeOH. These purified peaks were freeze-dried overnight, and the resulting solid was redissolved in a minimal volume (ca. 200 µL) of 50% aqueous MeOH, containing 1% NH3 for negative ion ESI-MS. ESI-MS Characterization of HPLC Product Peaks. The identities of the product peaks from HPLC separation were determined by ESI-MS using a VG Platform mass spectrometer (Fisons, NSW, Australia). The samples were delivered by a syringe pump (Harvard Apparatus 22) with a flow rate of 10 µL min-1 with 50 µL of sample used for analysis. A dry N2 bath gas at atmospheric pressure was employed to assist evaporation of the electrospray droplets. A negative electrospray mode was used with the following settings: capillary potential, 3.0 kV; chicane counter potential, 0.5 V; cone potential, 25 V; gain, 10; acquisition time, 4 min. The ESI-MS spectrum for the product peak with a retention time of 21.4 min was consistent with N-acetyl-BrTyr (m/z 299.8 and 301.8, intensity ratio of 1:1), and that at 38.0 min was consistent with N-acetyl-Br2-Tyr (m/z 377.7, 379.7, and 381.7, intensity ratio of 1:2:1).
Biochemistry, Vol. 43, No. 16, 2004 4801 Computational Modeling of HOBr ReactiVity. The computational modeling studies of HOBr with HSA and plasma amino acids were performed with Specfit software. A series of individual reactions describing the full reactivity of HSA, or the plasma amino acid composition, were used as the model as described previously for HOCl (6). The software was allowed to undergo 100 iterations to yield predicted reactant and product concentrations at time intervals where complete consumption of HOBr had occurred. RESULTS Kinetics of HOBr Reaction with Backbone Amide Groups. The kinetics for the reaction of HOBr (1.0 mM) with cyclo(Gly)2 (1.2-4.9 mM; equivalent to 2.4-9.8 mM amide) were investigated at 22 °C. Over 5 s a species with an absorbance band extending from 320 nm into the UV region was formed. The spectral changes are consistent with those observed for the analogous reaction with HOCl (6) and are attributed to the formation of cyclo(Gly)2 bromamide. The data were fitted by a simple mechanism [HOBr + cyclo(Gly)2 f bromamide], and Table 1 gives the resulting second-order rate constant. The experiments were repeated at 37 °C, and the second-order rate constant (Table 1) was 3.2 times greater than that at 22 °C. Experiments with HOBr prepared with increasing Br- excesses gave identical kinetics, suggesting that HOBr/-OBr, and not Br2, are the major species responsible for the observed reactions. Studies with cyclo(Ala)2, cyclo(Ser)2, and cyclo(Asp)2 yielded similar spectral changes, and the second-order rate constants are shown in Table 1. Reaction of HOBr (500 µM) with N-acetyl-Ala-OMe (2.5-10.0 mM) gave similar spectral changes to the cyclic dipeptides though over a longer time period (6 min). The resulting lower second-order rate constant (Table 1) is consistent with the analogous experiments with HOCl (6). A slow decay of the bromamide was observed over a period of 30 min, but the depletion was too small (typically
