What Are the Plasma Targets of the Oxidant Hypochlorous Acid? A Kinetic Modeling Approach David I. Pattison,* ,† Clare L. Hawkins, † and Michael J. Davies The Heart Research Institute, 114 Pyrmont Bridge Road, Camperdown, Sydney, NSW 2050, Australia ReceiVed October 8, 2008 Myeloperoxidase (MPO) is a heme enzyme, released by activated leukocytes at sites of inflammation, which catalyzes the formation of the potent oxidant, hypochlorous acid (HOCl), from H 2 O 2 . HOCl is a key component of the inflammatory response and is bactericidal but has been linked with several human pathologies as a result of damage to host tissue. Elevated plasma MPO levels are a strong independent risk factor, and predictor of outcomes, for cardiovascular disease. Rate constants for reaction of HOCl with individual biological targets and the products of these reactions have been determined, but the targets of HOCl in complex biological fluids such as plasma are incompletely defined. In this study, rate constants (M -1 s -1 ) for the reactions of ascorbate with HOCl (ca. 6 × 10 6 ) and imidazole chloramine (7.7 × 10 4 ) have been determined to supplement known kinetic parameters. HOCl-mediated oxidation of the major plasma protein, albumin, was investigated both experimentally and computationally; these approaches provide consistent data. The computational studies were extended to examine the fate of HOCl in plasma. The model predicts that plasma proteins consume the majority of HOCl with limited damage to other materials. Ascorbate or R-tocopherol, even at the levels achieved in human supplementation studies, do not attenuate these reactions. In contrast, elevated levels of thiocyanate ions (SCN - ), as detected in heavy smokers, can modulate HOCl-mediated reactions as a result of the formation of the highly specific oxidant hypothiocyanous acid (HOSCN). These observations support the hypothesis that MPO-generated HOSCN is a key agent in smoking-enhanced atherosclerosis. Introduction Myeloperoxidase (MPO) 1 plays a key role in the immune defense against invading pathogens (1), but it has become apparent that collateral damage to host tissues is a key event in a number of human pathologies linked with inflammation (reviewed in refs 2-5). Thus, elevated plasma MPO levels are a significant, independent risk factor for cardiovascular disease (6) and a strong predictor of health outcomes, for example, following myocardial infarction (7). MPO is a heme enzyme that catalyzes the reaction of H 2 O 2 [produced via the respiratory burst of leukocytes, as well as other cells (8)] with physiological concentrations of halide and pseudohalide ions (1). Thus, Cl - is converted to hypochlorous acid (HOCl), and Br - and SCN - ions are oxidized to hypo- bromous (HOBr) and hypothiocyanous (HOSCN) acid, respec- tively (2, 9). At physiological (plasma) concentrations of halide ions, the production of HOCl and HOSCN accounts for ca. 45 and 50%, respectively, of the H 2 O 2 consumed by MPO, with the remaining H 2 O 2 used to convert Br - to HOBr (9). HOCl, HOBr, and HOSCN possess potent bactericidal (HOCl, HOBr) and bacteriostatic (HOSCN) properties (2, 10). However, MPO- derived oxidants can initiate host tissue damage, with this having been detected in a wide range of inflammatory diseases including atherosclerosis, kidney disease, asthma, cystic fibrosis, and neurodegenerative disorders (reviewed in refs 2-5). Whether oxidants cause these diseases, as opposed to merely being a consequence of tissue inflammation, remains to be definitively established, but in at least some of these cases, the strong correlation between plasma MPO levels and disease incidence and outcomes implies a causative role. Thus, it is likely that elevated levels of HOCl are generated in human plasma and extracellular fluids, particularly at sites of inflammation. A number of studies have investigated the effects of HOCl formation in plasma by, for example, the quantification of end points of HOCl-mediated damage such as the extent of thiol or antioxidant consumption and protein cross-linking/aggregation (11-14). Other studies have quantified amine (e.g., terminal and side-chain amino groups of free amino acids and proteins) conversion to chloramines (RNHCl species), which are mod- erately stable, mild oxidizing agents (reviewed in ref 2). The breakdown products of chloramines, such as oxo-aldehydes (15, 16) and radical intermediates (11, 14), have also been quantified. However, limited data have been obtained on the speciation of such damage and particularly which of the multitude of components of plasma are the major targets of initial HOCl reaction (e.g., protein vs lipid vs free amino acids vs antioxidants or other components) and the sites of final damage [cf. evidence for chlorine transfer reactions between initial chloramines and other species (17-21)]. Recent studies have provided a wealth of rate constants for HOCl and chloramine reactions (reviewed in ref 22) that have allowed the development of computational kinetic models that predict the sites of HOCl-mediated damage to lipoproteins, proteoglycans, * To whom correspondence should be addressed. Tel: +61-2-8208-8900. Fax: +61-2-9565-5584. E-mail: pattisond@hri.org.au. † These authors contributed equally to the work presented in this manuscript. 1 Abbreviations: Cl-Tyr, 3-chlorotyrosine; DTNB, 5,5′-dithiobis-(2- nitrobenzoic acid); HisCl, the imidazole chloramine of the His side chain; HOCl, the equilibrium mixture of hypochlorous acid and its anion, OCl - , at physiological pH 7.4; IAACl, the imidazole chloramine of 4-imida- zoleacetic acid; LysCl, chloramine of the Lys side chain; MetS(O), methionine sulfoxide; MPO, myeloperoxidase; Tau, taurine; TauCl, chloram- ine on the amine group of Tau; TCA, trichloroacetic acid; termCl, chloramine at the R-amino group of a free amino acid or the N terminus of a protein; TNB, 5-thio-2-nitrobenzoic acid. Chem. Res. Toxicol. 2009, 22, 807–817 807 10.1021/tx800372d CCC: $40.75  2009 American Chemical Society Published on Web 03/27/2009