Reaction Of HOCl Research Articles

Computational Studies on Metathetical and Redox Processes of HOCl in Gas Phase. III. Its Self-Reaction and Interactions with HNOx (x = 1−3)

The gas-phase redox reactions of HOCl with its self and HNO(x) (x = 1-3) have been studied theoretically by ab initio quantum chemical and statistical mechanical theories. The structures of reactants, intermediate complexes, products, and transition states were optimized at the MPW1PW91/6-311+G(3df,2p) level of theory. The potential energy surface of each reaction was refined at the CCSD(T)/6-311+G(3df,2p) level of theory. The most favorable products are predicted to be ClClO + H(2)O and ClOCl + H(2)O for the HOCl self-reaction (A), H(2)O + Cl + NO for the HOCl + HNO reaction (B), H(2)O + ClNO(2) for the HOCl + HONO-t reaction (C), H(2)O + cis-ClONO for the HOCl + HONO-c reaction (D). For the HOCl + HONO(2) reaction (E), only one dehydration reaction channel was considered to produce H(2)O + ClONO(2). The rate constants of all above five reactions have been predicted at 300-3000 K by the VTST/RRKM theory. The calculation shows that the theoretical rate constants are within the upper limits of experimental results. In addition, we calculated the equilibrium constant for the Cl(2)O + H(2)O --> HOCl + HOCl reaction, which is also in reasonable agreement with experimental data within the error of the available experimental enthalpy change.

Reactive Sulfur Species: Kinetics and Mechanism of the Oxidation of Aryl Sulfinates with Hypochlorous Acid

The mechanism of oxidation of ArSO(2)(-) (PhSO(2)(-) and 5-sulfinato-2-nitrobenzoic acid = TNBO(2)(1-/2-)) with HOCl/OCl(-) has been investigated using the kinetic method. In contrast to previous reports for PhSO(2)(-) (for which it was suggested that OCl(-) and not HOCl was the reactant), the reaction proceeds through a conventional pathway: nucleophilic attack by ArSO(2)(-) on HOCl with concomitant Cl(+) transfer to give a sulfonyl chloride intermediate (ArSO(2)Cl), which we have identified spectrophotometrically. Remarkably, the rate constant for the reaction of HOCl with ArSO(2)(-) is on the order of 10(9) M(-1) s(-1), larger than the rate constants for corresponding thiolates, and is nearly diffusion-controlled. In contrast, the rate constant for the reaction of OCl(-) with ArSO(2)(-) is approximately 7 orders of magnitude smaller.

PH-Dependent Configurations of a 5-Chlorouracil-Guanine Base Pair

Hypochlorous acid (HOCl) from activated neutrophils at sites of inflammation can react with and damage biological molecules, including nucleic acids. The reaction of HOCl with cytosine analogues can generate multiple products, including 5-chlorouracil (ClU). In this paper, we have constructed oligonucleotides containing ClU paired opposite guanine (ClU-G). Melting studies indicate that oligonucleotide duplexes containing the ClU-G mispair are substantially less stable than those containing a ClU-A base pair. The melting temperature of the ClU-G mispair is not experimentally distinguishable from that of a T-G pair. NMR studies indicate that the ClU-G base pair adopts a wobble geometry at neutral pH, similar to a T-G mispair. The exchangeable protons of the ClU-G mispair broaden rapidly with an increase in temperature, indicating that the ClU-G mispair is less stable and opens more easily than the surrounding adjacent base pairs. Unlike the ClU-A base pair studied previously [Theruvathu, J. A., et al. (2009) Biochemistry 48, 7539-7546], the ClU-G mispair undergoes a pH-dependent structural change, assuming an ionized base pair configuration that approximates a Watson-Crick base pair at higher pH. Ionization of ClU in a DNA template could promote mispair formation and mutation, in accord with previous studies on other 5-halouracil analogues. The electron-withdrawing 5-chloro substituent facilitates ionization of the ClU N3 proton, promoting mispair formation, but it also renders the glycosidic bond susceptible to base cleavage by DNA repair glycosylases.

Computational Studies on Metathetical and Redox Processes of HOCl in the Gas Phase: (II) Reactions with ClOx (x = 1−4)

The reactions of HOCl + ClO(x) (x = 1-4) have been studied theoretically by ab initio quantum chemical and statistical mechanical methods. The structures of reactants, intermediates, products, and transition states were optimized at the MPW1PW91/6-311+G(3df,2p) level of theory, and the potential energy surface of each reaction was refined at the G2M and CCSD(T)/6-311+G(3df,2p) levels of theory. The most favorable reaction channels are predicted to be Cl-abstraction in HOCl + ClO with a barrier of 18.5 kcal/mol and H abstraction in HOCl + OClO with a barrier of 23.9 kcal/mol. In the HOCl + ClO(3) reaction both processes can occur; the barriers of Cl and H abstraction are 16.4 and 17.1 kcal/mol, respectively. In the HOCl + ClO(4) reaction, the H abstraction transition state lies below that of the reactants by 1.4 kcal/mol. The rate constants for all low barrier channels have been calculated in the temperature range 200-3000 K by statistical theory. In addition, the rate constant for the reverse of the HOCl + ClO reaction, Cl(2)O + OH --> HOCl + ClO, has been predicted; the result is in good agreement with the bulk of available experimental data.

Ametryn degradation by aqueous chlorine: Kinetics and reaction influences

The chemical oxidation of the herbicide ametryn was investigated by aqueous chlorination between pH 4 and 10 at a temperature of 25 °C. Ametryn was found to react very rapidly with aqueous chlorine. The reaction kinetics can be well described by a second-order kinetic model. The apparent second-order rate constants are greater than 5 × 10 2 M −1 s −1 under acidic and neutral conditions. The reaction proceeds much more slowly under alkaline conditions. The predominant reactions were found to be the reactions of HOCl with neutral ametryn and the charged ametryn, with rate constants equal to 7.22 × 10 2 and 1.58 × 10 3 M −1 s −1, respectively. The ametryn degradation rate increases with addition of bromide and decreases with addition of ammonia during the chlorination process. Based on elementary chemical reactions, a kinetic model of ametryn degradation by chlorination in the presence of bromide or ammonia ion was also developed. By employing this model, we estimate that the rate constants for the reactions of HOBr with neutral ametryn and charged ametryn were 9.07 × 10 3 and 3.54 × 10 6 M −1 s −1, respectively. These values are 10- to 10 3-fold higher than those of HOCl, suggesting that the presence of bromine species during chlorination could significantly accelerate ametryn degradation.

Differential efficiency of lysophospholipid markers for oxidative stress

Inflammatory liver diseases are associated with marked oxidative stress. At inflammatory loci, hypochlorous acid (HOCl) is generated by myeloperoxidase. HOCl reacts with a variety of molecules and induces the formation of lysophosphatidylcholine (LPC) from polyunsaturated phosphatidylcholine (PC). Since liver tissue contains huge amounts of polyunsaturated PC the arising of enhanced LPC concentrations could be expected and was experimentally verified in various species. Surprisingly, further lysophospholipids (LPL) could not be detected, although human liver contains many further polyunsaturated phospholipid (PL) classes. Therefore, four different isolated PL classes were investigated regarding the extent of LPL generation under the influence of HOCl. Using MALDI‐TOF mass spectrometry (MS) PC could be identified as the only PL leading to LPC, whereas phosphatidylglycerol, phosphatidylserine and phosphatidylethanolamine led exclusively to chlorohydrins by the reaction of HOCl with the double bonds of their unsaturated fatty acyl residues. Thus, LPC represents a useful biomarker of oxidative stress and inflammation that can be easily determined by MS ‐ even in complex mixtures.

What Are the Plasma Targets of the Oxidant Hypochlorous Acid? A Kinetic Modeling Approach

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 H2O2. 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 x 10(6)) and imidazole chloramine (7.7 x 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 alpha-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.

Effect of Chloride Ion on the Kinetics and Mechanism of the Reaction between Chlorite Ion and Hypochlorous Acid

The effect of chloride ion on the chlorine dioxide formation in the ClO 2 (-)-HOCl reaction was studied by following .ClO 2 concentration spectrophotometrically at pH 5-6 in 0.5 M sodium acetate. On the basis of the earlier experimental data collected without initially added chloride and on new experiments, the earlier kinetic model was modified and extended to interpret the two series of experiments together. It was found that the chloride ion significantly increases the initial rate of .ClO 2 formation. At the same time, the .ClO 2 yield is increased in HOCl but decreased in ClO 2 (-) excess by the increase of the chloride ion concentration. The two-step hydrolysis of dissolved chlorine through Cl 2 + H 2O left harpoon over right harpoon Cl 2OH (-) + H (+) and Cl 2OH (-) left harpoon over right harpoon HOCl + Cl (-) and the increased reactivity of Cl 2OH (-) compared to HOCl are proposed to explain these phenomena. It is reinforced that the hydrolysis of the transient Cl 2O 2 takes place through a HOCl-catalyzed step instead of the spontaneous hydrolysis. A seven-step kinetic model with six rate parameters (constants and/or ratio of constants) is proposed on the basis of the rigorous least-squares fitting of the parameters simultaneously to 129 absorbance versus time curves measured up to approximately 90% conversion. The advantage of this method of evaluation is briefly outlined.

Photooxidation of dicarboxylic acids—Part I: Effects of inorganic ions on degradation of azelaic acid

In this paper, the first of a two-part series, effects of chloride, sulfate, and nitrate ions and pH on photooxidation of azelaic acid were investigated in an aqueous system. Nitrate ions play the major role in accelerating photooxidation of azelaic acid by increasing OH concentration, while chloride ions consume OH concentration and retard photooxidation rates. In inorganic mixtures, a nitrate-to-chloride molar ratio of >1.5 accelerated photooxidation of azelaic acid indicating the dominant role of nitrate. Substantial inhibition effects of chloride on photooxidation of azelaic acid were demonstrated at a nitrate-to-chloride molar ratio <0.3. Nitrate and chloride are interrelated in affecting photooxidation of azelaic acid as photolysis of nitrate would significantly consume H +, retarding reaction of HOCl − with H +, and consequently decreasing OH-chloride reaction. pH affects photooxidation of C 2–C 9 dicarboxylic acids (DCAs) in two ways: C 2–C 4 dicarboxylates exhibit substantially higher degradation rates than their parent DCAs, while C 5–C 9 dicarboxylates demonstrate degradation rates similar to their parent DCAs.

Hypochlorous Acid-Mediated Protein Oxidation: How Important Are Chloramine Transfer Reactions and Protein Tertiary Structure?

Hypochlorous acid (HOCl) is a powerful oxidant generated from H2O2 and Cl- by the heme enzyme myeloperoxidase, which is released from activated leukocytes. HOCl possesses potent antibacterial properties, but excessive production can lead to host tissue damage that occurs in numerous human pathologies. As proteins and amino acids are highly abundant in vivo and react rapidly with HOCl, they are likely to be major targets for HOCl. In this study, two small globular proteins, lysozyme and insulin, have been oxidized with increasing excesses of HOCl to determine whether the pattern of HOCl-mediated amino acid consumption is consistent with reported kinetic data for isolated amino acids and model compounds. Identical experiments have been carried out with mixtures of N-acetyl amino acids (to prevent reaction at the alpha-amino groups) that mimic the protein composition to examine the role of protein structure on reactivity. The results indicate that tertiary structure facilitates secondary chlorine transfer reactions of chloramines formed on His and Lys side chains. In light of these data, second-order rate constants for reactions of Lys side chain and Gly chloramines with Trp side chains and disulfide bonds have been determined, together with those for further oxidation of Met sulfoxide by HOCl and His side chain chloramines. Computational kinetic models incorporating these additional rate constants closely predict the experimentally observed amino acid consumption. These studies provide insight into the roles of chloramine formation and three-dimensional structure on the reactions of HOCl with isolated proteins and demonstrate that kinetic models can predict the outcome of HOCl-mediated protein oxidation.

Influence of chloride on modification of unsaturated phosphatidylcholines by the myeloperoxidase/hydrogen peroxide/bromide system

The leukocyte enzyme myeloperoxidase (MPO) is capable of catalyzing the oxidation of chloride and bromide ions, at physiological concentrations of these substrates, by hydrogen peroxide, generating hypochlorous acid (HOCl) and hypobromous acid (HOBr), respectively. Our previous results showed that the hypohalous acids formed react with double bonds in phosphatidylcholines (PCs) to produce chloro- and bromohydrins. Lysophosphatidylcholine (lyso-PC) is additionally formed in PCs with two or more double bonds. This study was conducted to determine the effect physiological chloride concentration (140 mM) has on the formation of bromohydrins and lyso-PC from unsaturated PC upon treatment with the myeloperoxidase/hydrogen peroxide/bromide (MPO/H 2O 2/Br −) system using physiological bromide concentrations (20–100 μM). The composition of reaction products was analyzed by matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS). With monounsaturated PC, we demonstrated that the rate and extent of mono-bromohydrin formation were higher in the samples with 140 mM chloride compared to those with no added chloride. Moreover, mono-bromohydrin came to be the major product and no mono-chlorohydrin was observed already at 60 μM bromide. We attributed these effects to the involvement of HOBr arising from the reaction of MPO-derived HOCl with bromide rather than to the exchange of bromide with chlorine atoms of chlorohydrins or direct formation of HOBr by MPO. The presence of chloride shifted the pH optimum for mono-bromohydrin formation (pH 5.0) toward neutral values, and a significant yield of mono-bromohydrin was detected at physiological pH values (7.0–7.4). For polyunsaturated PC, chloride enhanced also lyso-PC production, the effect being pronounced at bromide concentrations below 40 μM. The results indicate that at physiological levels of chloride and bromide, chloride promotes MPO-mediated formation of bromohydrins and lyso-PC in unsaturated phospholipids.

Chlorine photolysis and subsequent OH radical production during UV treatment of chlorinated water

The photodegradation of chlorine-based disinfectants NH 2Cl, HOCl, and OCl − under UV irradiation from low- (LP) and medium-pressure (MP) Hg lamps was studied. The quantum yields of aqueous chlorine and chloramine under 254 nm (LP UV) irradiation were greater than 1.2 mol Es −1 for free chlorine in the pH range of 4–10 and 0.4 mol Es −1 for monochloramine at pH 9. Quantum yields for MP (200–350 nm) ranged from 1.2 to 1.7 mol Es −1 at neutral and basic pH to 3.7 mol Es −1 at pH 4 for free chlorine, and 0.8 mol Es −1 for monochloramine. Degradation of free chlorine was enhanced under acidic water conditions, but water quality negatively impacted the MP Hg lamp degradation of free chlorine, compared to the LP UV source. The production of hydroxyl radical via chlorine photolysis was assessed along with the rate of reaction between OH and HOCl using radical scavengers (parachlorobenzoic acid and nitrobenzene) in chlorinated solutions at pH 4. The quantum yield of OH radical production from HOCl at 254 nm was found to be 1.4 mol Es −1, while the reaction of HOCl with OH radical was measured as 8.5×10 4 M −1 s −1. NH 2Cl was relatively stable in all irradiated solutions, with <0.3 mg L −1 increase in nitrate following a UV dose of 1000 mJ cm −2. For water treatment plants, no significant changes in chlorine concentration would be expected under typical pH levels and UV doses; however, the formation of OH could have implications for chlorinated byproducts or decay of unwanted chemical contaminants.

Automatic in Vitro Determination of Hypochlorous Acid Scavenging Capacity Exploiting Multisyringe Flow Injection Analysis and Chemiluminescence

In the present work, a chemiluminometric automatic flow methodology for the in vitro determination of hypochlorous acid scavenging capacity, under pH and concentration conditions similar to those found in vivo, is proposed. As the pH found in physiological conditions (7.4) and the pH required for the chemiluminescence detection reaction (>10) are different, the multisyringe flow injection analysis features were exploited to perform the in-line reaction of HOCl and the scavenger molecule at physiological pH prior to reaction of the remaining HOCl with luminol at alkaline conditions. These two reactions were carried out in about 3 s, allowing the determination of fast reacting antioxidants, in a time frame closer to in vivo generation of HOCl when compared to previously described methods. The developed method was applied to nonsteroidal antiinflammatory drugs of different chemical families, and positive controls (cysteine, gallic acid, lipoic acid). The HOCl scavenging capacity was evaluated at pH 7.4 and 10.0; different results were found for oxicam derivatives, providing evidence that the pH of in vitro methods should be carefully selected to allow assumptions about putative in vivo effects.

Chlorination by a Long-Lived Intermediate in the Mechanism of Flavin-Dependent Halogenases,

The flavin-dependent halogenase RebH catalyzes the formation of 7-chlorotryptophan as the initial step in the biosynthesis of antitumor agent rebeccamycin. The reaction of FADH2, Cl-, and O2 in the active site generates the powerful oxidant HOCl, which was presumed to carry out the chlorination reaction. Herein, we demonstrate the formation of a long-lived chlorinating intermediate (t1/2 = 63 h at 4 degrees C) when RebH, FADH2, Cl-, and O2 react in the absence of substrate tryptophan. This intermediate remained on the enzyme after removal of FAD and transferred chlorine to tryptophan with kinetically competent rates. The identity of this intermediate is suggested by the X-ray crystal structure of RebH, which revealed an active site Lys79 located in a central position between flavin and tryptophan binding sites and just 4.1 A above C7 of tryptophan. The chlorinating species is proposed to be a Lys-epsilonNH-Cl (lysine chloramine) from reaction of enzyme-generated HOCl with the active site Lys79. This covalent enzyme chloramine likely plays a key role in directing regiospecific chlorination of substrate in this important class of biosynthetic enzymes.

Study by XPS of the chlorination of proteins aggregated onto tin dioxide during electrochemical production of hypochlorous acid

In solution, hypochlorous acid (HOCl) reacts with organic matter and notably with protein side-chains. In this study, HOCl was produced by an electrochemical way, by oxidation of chloride ions at a transparent tin dioxide electrode in the presence of a protein, the bovine serum albumin (BSA). A thick irregular layer is formed at the electrode when HOCl is produced at the SnO 2 surface. Indeed, SEM analyses show that an important deposit is formed during the anodic polarization of SnO 2 in the presence of chloride ions and proteins. Actually, two phenomena take place on the one hand the chlorination of the proteins due to the reaction of HOCl with some protein side-chains and on the other hand the aggregation of proteins onto the SnO 2 surface. The present X-ray photoelectron spectroscopy study points out the cross-linking of BSA molecules via formation of inter molecular sulfonamide groups. It also shows that the BSA chlorination is due on the one hand to the formation of sulfonyl chloride groups (–SO 2Cl) and on the other hand to formation of chloramine groups ( N–Cl). The Cl2p and S2p photo-peak intensities allowed us to quantify the chloramines. It is found that, one BSA entity immobilized onto the SnO 2 surface contains about 50 chloramine groups.

Hypochlorous Acid-Derived Modification of Phospholipids: Characterization of Aminophospholipids as Regulatory Molecules for Lipid Peroxidation

Hypochlorous acid (HOCl), an inflammatory oxidant derived from neutrophil myeloperoxidase, can chlorinate cytosolic proteins and nuclear DNA bases of target cells by passing through the cell membrane. However, little is known about the consequences of HOCl-derived modification of cell membrane components, including phospholipids. In this study, we characterize the reaction of HOCl with phospholipid molecules and found that aminophospholipids are the key molecules that chemically regulate lipid peroxidation. Upon incubation with HOCl, the peroxidation of egg yolk phosphatidylcholine was significantly enhanced in the presence of phosphatidylethanolamine (PE). In contrast, the peroxidation was significantly inhibited in the presence of phosphatidylserine (PS). On the basis of mass spectrometric and electron paramagnetic resonance characterization, the initiator of the peroxidation was identified as the nitrogen-centered radical originating from PE-derived chloramines, especially N,N-dichlorinated PE, a major product in the HOCl-modified PE. Although PS was also chlorinated upon reaction with HOCl, the formed chloramine rapidly decomposed to phosphatidylglycolaldehyde, a novel class of lipid aldehyde. Formation of phosphatidylglycolaldehyde was also confirmed in the porcine brain PS and erythrocyte cell membrane ghost exposed to HOCl. These results provide a novel mechanism for the HOCl-induced oxidative damage and its endogenous protection in the cell membrane at the site of inflammation.

Evidence for Rapid Inter- and Intramolecular Chlorine Transfer Reactions of Histamine and Carnosine Chloramines: Implications for the Prevention of Hypochlorous-Acid-Mediated Damage

Hypochlorous acid (HOCl) is a powerful oxidant generated from H(2)O(2) and Cl(-) by the heme enzyme myeloperoxidase, which is released from activated leukocytes. HOCl possesses potent antibacterial properties, but excessive production can lead to host tissue damage that is implicated in a wide range of human diseases (e.g., atherosclerosis). Histamine and carnosine have been proposed as protective agents against such damage. However, as recent studies have shown that histidine-containing compounds readily form imidazole chloramines that can rapidly chlorinate other targets, it was hypothesized that similar reactions may occur with histamine and carnosine, leading to propagation, rather than prevention, of HOCl-mediated damage. In this study, the reactions of HOCl with histamine, histidine, carnosine, and other compounds containing imidazole and free amine sites were examined. In all cases, rapid formation (k, 1.6 x 10(5) M(-)(1) s(-)(1)) of imidazole chloramines was observed, followed by chlorine transfer to yield more stable, primary chloramines (R-NHCl). The rates of most of these secondary reactions are dependent upon substrate concentrations, consistent with intermolecular mechanisms (k, 10(3)-10(4) M(-)(1) s(-)(1)). However, for carnosine, the imidazole chloramine transfer rates are independent of the concentration, indicative of intramolecular processes (k, 0.6 s(-)(1)). High-performance liquid chromatography studies show that in all cases the resultant R-NHCl species can slowly chlorinate N-alpha-acetyl-Tyr. Thus, the current data indicate that the chloramines formed on the imidazole and free amine groups of these compounds can oxidize other target molecules but with limited efficiency, suggesting that histamine and particularly carnosine may be able to limit HOCl-mediated oxidation in vivo.

Thiocyanate Is an Efficient Endogenous Scavenger of the Phagocytic Killing Agent Hypobromous Acid

Second-order rate constants for the reaction of HOBr/OBr- (a putative killing agent of eosinophils and a reactive oxygen species that is implicated in mutagenesis and in human inflammatory diseases) with SCN- (an endogenous species in human physiologic fluids) are determined by stopped-flow spectroscopy. The proposed mechanism includes parallel pathways with Br+ transfer to SCN- by general acid catalysis and by direct reaction with HOBr. HOBr reacts with SCN- with a second-order rate constant (2.3 x 10(9) M(-1) s(-1)) that is 2 orders of magnitude larger than that previously measured for the reaction of HOCl with SCN- (2.3 x 10(7) M(-1) s(-1)), and very close to the diffusion limit. In contrast to OCl-, OBr- exhibits a measurable rate of reaction with SCN- (3.8 x 10(4) M(-1) s(-1)). On a molar basis, SCN- is the most effective scavenger of HOBr to be reported to date (200 times more effective than cysteine and 650 times more effective than methionine). Computational models suggest that SCN- is competitive with respect to other scavengers at physiologically relevant concentrations, which leads us to propose it may limit the lifetime of HOBr and its propensity to inflict host tissue damage during inflammatory response, especially during eosinophilia. Furthermore, the product of the nonenzymatic reaction of HOBr and SCN-, hypothiocyanite (OSCN-), is an effective antimicrobial that is relatively innocuous toward mammalian cell lines. Since one of the principal charges of eosinophil cells is to clear extracellular parasites via nonphagocytic mechanisms that involve degranulation of eosinophil peroxidase (EPO, the principal mammalian enzyme that produces HOBr), a larger role for OSCN- is suggested for parasitic infection.

Oxidation of heparan sulphate by hypochlorite: role of N-chloro derivatives and dichloramine-dependent fragmentation

Activated phagocytes release the haem enzyme MPO (myeloperoxidase) and produce superoxide radicals and H2O2 via an oxidative burst. MPO uses H2O2 and Cl- to form HOCl, the physiological mixture of hypochlorous acid and its anion present at pH 7.4. As MPO binds to glycosaminoglycans, oxidation of extracellular matrix and cell surfaces by HOCl may be localized to these materials. However, the reactions of HOCl with glycosaminoglycans are poorly characterized. The GlcNAc (N-acetylglucosamine), GlcNSO3 (glucosamine-N-sulphate) and GlcNH2 [(N-unsubstituted) glucosamine] residues of heparan sulphate are potential targets for HOCl. It is shown here that HOCl reacts with each of these residues to generate N-chloro derivatives, and the absolute rate constants for these reactions have been determined. Reaction at GlcNH2 residues yields chloramines and, subsequently, dichloramines with markedly slower rates, k2 approximately 3.1x10(5) and 9 M(-1) x s(-1) (at 37 degrees C) respectively. Reaction at GlcNSO3 and GlcNAc residues yields N-chlorosulphonamides and chloramides with k2 approximately 0.05 and 0.01 M(-1) x s(-1) (at 37 degrees C) respectively. The corresponding monosaccharides display a similar pattern of reactivity. Decay of the polymer-derived chloramines, N-chlorosulphonamides and chloramides is slow at 37 degrees C and does not result in major structural changes. In contrast, dichloramine decay is rapid at 37 degrees C and results in fragmentation of the polymer backbone. Computational modelling of the reaction of HOCl with heparan sulphate proteoglycans (glypican-1 and perlecan) predicts that the GlcNH2 residues of heparan sulphate are major sites of attack. These results suggest that HOCl may be an important mediator of damage to glycosaminoglycans and proteoglycans at inflammatory foci.

Inactivation of Protease Inhibitors and Lysozyme by Hypochlorous Acid: Role of Side-Chain Oxidation and Protein Unfolding in Loss of Biological Function

Excessive or misplaced activation of leukocytes causes host tissue damage which has been implicated in diseases such as atherosclerosis and chronic inflammation. This may arise via either the generation of oxidants such as hypochlorous acid (HOCl) by the heme enzyme myeloperoxidase, the action of released enzymes including lysozyme and proteases, or a combination of these two activities. Thus, oxidant-mediated inactivation of protease inhibitors that modulate tissue proteolysis by the released enzymes may exacerbate protease-induced degradation of host tissue. The role of myeloperoxidase-derived oxidants, such as HOCl, in the inactivation of Kunitz-type inhibitors and lysozyme is not well-characterized and is the subject of the current study. Exposure of both trypsin inhibitor and lysozyme to low molar excesses of HOCl compared to protein is shown to result in loss of function. With trypsin inhibitor, this loss of activity is associated with the selective oxidation of Trp, Tyr, and His residues, which results in protein unfolding and the disruption of complex formation with active trypsin. Oxidation of Met residues, a major target for HOCl, or the active site Arg, does not appear to play a key role in this loss of activity. In contrast, with lysozyme, oxidation of Met residues to Met sulfoxide appears to be the major process resulting in loss of enzyme activity. With both proteins, inactivation occurs in a time-dependent manner, consistent with both direct oxidation by HOCl and secondary reactions of protein chloramines formed from amine groups (e.g., from Lys and His) playing a role in loss of activity.