Hypochlorous acid-modified low-density lipoprotein inactivates the lysosomal protease cathepsin B: protection by ascorbic and lipoic acids

Unregulated uptake of oxidized LDL by the scavenger receptor(s) of macrophages is thought to be an early event in atherosclerotic lesion development. Accumulation of oxidized LDL within macrophages may result from resistance of the modified LDL to enzymatic hydrolysis or from direct inactivation of lysosomal enzymes by reactive LDL-associated moieties. Since HOCl-modified LDL has been detected in vivo, the effects of HOCI-modified LDL on the activities of the cysteine protease cathepsin B and the aspartyl protease cathepsin D were investigated. LDL (0.5 mg protein/ml), which had been exposed to HOCl (25-200 microM), caused rapid dose-dependent inactivation of cathepsin B, but not of cathepsin D. Exposure of LDL to HOCl results primarily in the formation of LDL-associated chloramines, and the model chloramine N(alpha)-acetyl-lysine chloramine also caused dose-dependent inactivation of cathepsin B. Incubation of HOCl-modified LDL with ascorbic and lipoic acids (25-200 microM) resulted in dose-dependent reduction of LDL-associated chloramines and concomitant protection against cathepsin B inactivation. Thus, the data indicate that HOCl-modified LDL inactivates cathepsin B by a chloramine-dependent mechanism, most likely via oxidation of the enzyme's critical cysteine residue. Furthermore, small molecule antioxidants, such as ascorbic and lipoic acids, may be able to inhibit this potentially pro-atherogenic process by scavenging LDL-associated chloramines.     

Taurine chloramine is more selective than hypochlorous acid at targeting critical cysteines and inactivating creatine kinase and glyceraldehyde-3-phosphate dehydrogenase

Hypochlorous acid (HOCl) and chloramines are produced by the neutrophil enzyme, myeloperoxidase. Both react readily with thiols, although chloramines differ from HOCl in discriminating between low molecular weight thiols on the basis of their pKa. Here, we have compared the reactivity of HOCl and taurine chloramine with thiol proteins by examining inactivation of creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). With both enzymes, loss of activity paralleled thiol loss. For CK both were complete at a 1:1 taurine chloramine:thiol mole ratio. For GAPDH each chloramine oxidized two thiols. Three times more HOCl than taurine chloramine was required for inactivation, indicating that HOCl is less thiol specific. Competition studies showed that thiols of CK were 4 times more reactive with taurine chloramine than thiols of GAPDH (rate constants of 1200 and 300 M-1s-1 respectively). These compare with 205 M-1s-1 for cysteine and are consistent with their lower pKa's. Both enzymes were equally susceptible to HOCl. GSH competed directly with the enzyme thiols for taurine chloramine and protected against oxidative inactivation. At lower GSH concentrations, mixed disulfides were formed. We propose that chloramines should preferentially attack proteins with low pKa thiols and this could be important in regulatory processes.     

Protein thiol oxidation and formation of S-glutathionylated cyclophilin A in cells exposed to chloramines and hypochlorous acid

Neutrophil oxidants, including the myeloperoxidase products, HOCl and chloramines, have been linked to endothelial dysfunction in inflammatory diseases such as atherosclerosis. As they react preferentially with sulfur centers, thiol proteins are likely to be cellular targets. Our objectives were to establish whether there is selective protein oxidation in vascular endothelial cells treated with HOCl or chloramines, and to identify sensitive proteins. Cells were treated with HOCl, glycine chloramine and monochloramine, reversibly oxidized cysteines were labeled and separated by 1D or 2D SDS-PAGE, and proteins were characterized by mass spectrometry. Selective protein oxidation was observed, with chloramines and HOCl causing more changes than H(2)O(2). Cyclophilin A was one of the most sensitive targets, particularly with glycine chloramine. Cyclophilin A was also oxidized in Jurkat T cells where its identity was confirmed using a knockout cell line. The product was a mixed disulfide with glutathione, with glutathionylation at Cys-161. Glyceraldehyde-3-phosphate dehydrogenase, peroxiredoxins and cofilin were also highly sensitive to HOCl/chloramines. Cyclophilins are becoming recognized as redox regulatory proteins, and glutathionylation is an important mechanism for redox regulation. Cells lacking Cyclophilin A showed more glutathionylation of other proteins than wild-type cells, suggesting that cyclophilin-regulated deglutathionylation could contribute to redox changes in HOCl/chloramine-exposed cells.     

Bilirubin scavenges chloramines and inhibits myeloperoxidase-induced protein/lipid oxidation in physiologically relevant hyperbilirubinemic serum

Hypochlorous acid (HOCl), an oxidant produced by myeloperoxidase (MPO), induces protein and lipid oxidation, which is implicated in the pathogenesis of atherosclerosis. Individuals with mildly elevated bilirubin concentrations (i.e., Gilbert syndrome; GS) are protected from atherosclerosis, cardiovascular disease, and related mortality. We aimed to investigate whether exogenous/endogenous unconjugated bilirubin (UCB), at physiological concentrations, can protect proteins/lipids from oxidation induced by reagent and enzymatically generated HOCl. Serum/plasma samples supplemented with exogenous UCB (≤250 µM) were assessed for their susceptibility to HOCl and MPO/H₂O₂/Cl⁻ oxidation, by measuring chloramine, protein carbonyl, and malondialdehyde (MDA) formation. Serum/plasma samples from hyperbilirubinemic Gunn rats and humans with GS were also exposed to MPO/H₂O₂/Cl⁻ to: (1) validate in vitro data and (2) determine the relevance of endogenously elevated UCB in preventing protein and lipid oxidation. Exogenous UCB dose-dependently (P<0.05) inhibited HOCl and MPO/H₂O₂/Cl⁻-induced chloramine formation. Albumin-bound UCB efficiently and specifically (3.9–125 µM; P<0.05) scavenged taurine, glycine, and N-α-acetyllysine chloramines. These results were translated into Gunn rat and GS serum/plasma, which showed significantly (P<0.01) reduced chloramine formation after MPO-induced oxidation. Protein carbonyl and MDA formation was also reduced after MPO oxidation in plasma supplemented with UCB (P<0.05; 25 and 50 µM, respectively). Significant inhibition of protein and lipid oxidation was demonstrated within the physiological range of UCB, providing a hypothetical link to protection from atherosclerosis in hyperbilirubinemic individuals. These data demonstrate a novel and physiologically relevant mechanism whereby UCB could inhibit protein and lipid modification by quenching chloramines induced by MPO-induced HOCl.     

IkappaB is a sensitive target for oxidation by cell-permeable chloramines: inhibition of NF-kappaB activity by glycine chloramine through methionine oxidation

Hypochlorous acid (HOCl) is produced by the neutrophil enzyme, myeloperoxidase, and reacts with amines to generate chloramines. These oxidants react readily with thiols and methionine and can affect cell-regulatory pathways. In the present study, we have investigated the ability of HOCl, glycine chloramine (Gly-Cl) and taurine chloramine (Tau-Cl) to oxidize IkappaBalpha, the inhibitor of NF-kappaB (nuclear factor kappaB), and to prevent activation of the NF-kappaB pathway in Jurkat cells. Glycine chloramine (Gly-Cl) and HOCl were permeable to the cells as determined by oxidation of intracellular GSH and inactivation of glyceraldehyde-3-phosphate dehydrogenase, whereas Tau-Cl showed no detectable cell permeability. Both Gly-Cl (20-200 muM) and HOCl (50 microM) caused oxidation of IkappaBalpha methionine, measured by a shift in electrophoretic mobility, when added to the cells in Hanks buffer. In contrast, a high concentration of Tau-Cl (1 mM) in Hanks buffer had no effect. However, Tau-Cl in full medium did modify IkappaBalpha. This we attribute to chlorine exchange with other amines in the medium to form more permeable chloramines. Oxidation by Gly-Cl prevented IkappaBalpha degradation in cells treated with TNFalpha (tumour necrosis factor alpha) and inhibited nuclear translocation of NF-kappaB. IkappaBalpha modification was reversed by methionine sulphoxide reductase, with both A and B forms required for complete reduction. Oxidized IkappaBalpha persisted intracellularly for up to 6 h. Reversion occurred in the presence of cycloheximide, but was prevented if thioredoxin reductase was inhibited, suggesting that it was due to endogenous methionine sulphoxide reductase activity. These results show that cell-permeable chloramines, either directly or when formed in medium, could regulate NF-kappaB activation via reversible IkappaBalpha oxidation.     

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.     

Chloramines and hypochlorous acid oxidize erythrocyte peroxiredoxin 2

Peroxiredoxin 2 (Prx2) is an abundant thiol protein that is readily oxidized in erythrocytes exposed to hydrogen peroxide. We investigated its reactivity in human erythrocytes with hypochlorous acid (HOCl) and chloramines, relevant oxidants in inflammation. Prx2 was oxidized to a disulfide-linked dimer by HOCl, glycine chloramine (GlyCl), and monochloramine (NH₂Cl) in a dose-dependent manner. In the absence of added glucose, Prx2 and GSH showed similar sensitivities. Second-order rate constants for the reactions of Prx2 with NH₂Cl and GlyCl were 1.5 × 10⁴ and 8 M⁻¹ s⁻¹, respectively. The NH₂Cl value is 10 times higher than that for GSH, whereas Prx2 is 30 times less sensitive than GSH to GlyCl. Thus, the relative sensitivity of Prx2 to GlyCl is greater in the erythrocyte. Oxidation of erythrocyte Prx2 and GSH was less in the presence of glucose, probably because of recycling. High doses of NH₂Cl resulted in incomplete regeneration of reduced Prx2, suggesting impairment of the recycling mechanism. Our results show that, although HOCl and chloramines are less selective than H₂O₂, they nevertheless oxidize Prx2. Exposure to these inflammatory oxidants will result in Prx2 oxidation and could compromise the erythrocyte's ability to resist damaging oxidative insult.     

Histamine chloramine reactivity with thiol compounds, ascorbate, and methionine and with intracellular glutathione

Histamine is stored in granules of mast cells and basophils and released by inflammatory mediators. It has the potential to intercept some of the HOCl generated by the neutrophil enzyme, myeloperoxidase, to produce histamine chloramine. We have measured rate constants for reactions of histamine chloramine with methionine, ascorbate, and GSH at pH 7.4, of 91 M(-1)s(-1), 195 M(-1)s(-1), and 721 M(-1)s(-1), respectively. With low molecular weight thiols, the reaction was with the thiolate and rates increased exponentially with decreasing thiol group pK(a). Comparing rate constants for different chloramines reacting with ascorbate or a particular thiol anion, these were higher when there was less negative charge in the vicinity of the chloramine group. Histamine chloramine was the most reactive among biologically relevant chloramines. Consumption of histamine chloramine and oxidation of intracellular GSH were examined for human fibroblasts. At nontoxic doses, GSH loss over 10 min was slightly greater than that with HOCl, but the cellular uptake of histamine chloramine was 5-10-fold less. With histamine chloramine, GSSG was a minor product and most of the GSH was converted to mixed disulfides with proteins. HOCl gave a different profile of GSH oxidation products, with significantly less GSSG and mixed disulfide formation. There was irreversible oxidation and losses to the medium, as observed with HOCl and other cell types. Thus, histamine chloramine shows high preference for thiols both in isolation and in cells, and in this respect is more selective than HOCl.     

Class B scavenger receptors CD36 and SR-BI are receptors for hypochlorite-modified low density lipoprotein

The presence of HOCl-modified epitopes inside and outside monocytes/macrophages and the presence of HOCl-modified apolipoprotein B in atherosclerotic lesions has initiated the present study to identify scavenger receptors that bind and internalize HOCl-low density lipoprotein (LDL). The uptake of HOCl-LDL by THP-1 macrophages was not saturable and led to cholesterol/cholesteryl ester accumulation. HOCl-LDL is not aggregated in culture medium, as measured by dynamic light scattering experiments, but internalization of HOCl-LDL could be inhibited in part by cytochalasin D, a microfilament disrupting agent. This indicates that HOCl-LDL is partially internalized by a pathway resembling phagocytosis-like internalization (in part by fluid-phase endocytosis) as measured with [14C]sucrose uptake. In contrast to uptake studies, binding of HOCl-LDL to THP-1 cells at 4 degrees C was specific and saturable, indicating that binding proteins and/or receptors are involved. Competition studies on THP-1 macrophages showed that HOCl-LDL does not compete for the uptake of acetylated LDL (a ligand to scavenger receptor class A) but strongly inhibits the uptake of copper-oxidized LDL (a ligand to CD36 and SR-BI). The binding specificity of HOCl-LDL to class B scavenger receptors could be demonstrated by Chinese hamster ovary cells overexpressing CD36 and SR-BI and specific blocking antibodies. The lipid moiety isolated from the HOCl-LDL particle did not compete for cell association of labeled HOCl-LDL to CD36 or SR-BI, suggesting that the protein moiety of HOCl-LDL is responsible for receptor recognition. Experiments with Chinese hamster ovary cells overexpressing scavenger receptor class A, type I, confirmed that LDL modified at physiologically relevant HOCl concentrations is not recognized by this receptor.     

A spectrophotometric assay for chlorine-containing compounds.

Determinations of hypochlorous acid and chloramine compounds are important in a number of areas. Several techniques are now available for such analyses, but most require unstable reagents and/or multiple steps in the analytical procedure. We have developed a simple, one-step spectrophotometric assay for reactive chlorine-containing compounds involving the oxidation of ascorbic acid by hypochlorous acid or chloramines. There is no interference from other nonhalide oxidants such as hydrogen peroxide or hypothiocyanous acid. Because small amounts of ascorbic acid will not damage biological materials, this method also allows continuous measurements of the generation of chlorine-containing compounds by activated neutrophils. This simple assay permits precise analysis of as little as 1 nmol of HOCl.     

Hypochlorite-modified low-density lipoprotein induces the apoptotic machinery in Jurkat T-cell lines

Myeloperoxidase is abundantly present in inflammatory diseases where activation of monocytes/macrophages and T-cell-mediated immune response occurs. The potent oxidant hypochlorous acid (HOCl), generated by the myeloperoxidase–H₂O₂–chloride system of activated phagocytes, converts low-density lipoprotein (LDL) into a proinflammatory lipoprotein particle. Here, we investigated the apoptotic effect of HOCl–LDL, an in vivo occurring LDL modification, on human T-cell lymphoblast-like Jurkat cells. Experiments revealed that HOCl–LDL, depending on the oxidant:lipoprotein molar ratio, induces apoptosis via activation of caspase-3, PARP cleavage and accumulation of reactive oxygen species. The absence of Fas-associated protein with death domain or caspase-8 in mutant cells did not prevent HOCl–LDL induced apoptosis. In contrast, overexpression of the anti-apoptotic Bcl-2 protein protects Jurkat cells against HOCl–LDL-induced apoptosis and prevents accumulation of reactive oxygen species. We conclude that HOCl–LDL-mediated apoptosis in Jurkat cells follows predominantly the intrinsic, mitochondrial pathway. Insitu experiments revealed that an antibody raised against HOCl–LDL recognized epitopes that colocalize both with myeloperoxidase and CD3-positive T-cells in human decidual tissue where local stimulation of the immune system occurs. We provide convincing evidence that formation of HOCl-modified (lipo)proteins generated by the myeloperoxidase–H₂O₂–chloride system contributes to apoptosis in T-cells.     

Cathepsin B Gene Disruption Induced Leishmania donovani Proteome Remodeling Implies Cathepsin B Role in Secretome Regulation

Leishmania cysteine proteases are potential vaccine candidates and drug targets. To study the role of cathepsin B cysteine protease, we have generated and characterized cathepsin B null mutant L. donovani parasites. L. donovani cathepsin B null mutants grow normally in culture, but they show significantly attenuated virulence inside macrophages. Quantitative proteome profiling of wild type and null mutant parasites indicates cathepsin B disruption induced remodeling of L. donovani proteome. We identified 83 modulated proteins, of which 65 are decreased and 18 are increased in the null mutant parasites, and 66% (55/83) of the modulated proteins are L. donovani secreted proteins. Proteins involved in oxidation-reduction (trypanothione reductase, peroxidoxins, tryparedoxin, cytochromes) and translation (ribosomal proteins) are among those decreased in the null mutant parasites, and most of these proteins belong to the same complex network of proteins. Our results imply virulence role of cathepsin B via regulation of Leishmania secreted proteins.     

Chlorination of N-acetyltyrosine with HOCl, chloramines, and myeloperoxidase-hydrogen peroxide-chloride system.

N-acetyl-L-tyrosine (N-acTyr), with the alpha amine residue blocked by acetylation, can mimic the reactivity of exposed tyrosyl residues incorporated into polypeptides. In this study chlorination of N-acTyr residue at positions 3 and 5 in reactions with NaOCl, chloramines and the myeloperoxidase (MPO)-H2O2-Cl- chlorinating system were invesigated. The reaction of N-acTyr with HOCl/OCl- depends on the reactant concentration ratio employed. At the OCl-/N-acTyr (molar) ratio 1:4 and pH 5.0 the chlorination reaction yield is about 96% and 3-chlorotyrosine is the predominant reaction product. At the OCl-/N-acTyr molar ratio 1:1.1 both 3-chlorotyrosine and 3,5-dichlorotyrosine are formed. The yield of tyrosine chlorination depends also on pH, amounting to 100% at pH 5.5, 91% at pH 4.5 and 66% at pH 3.0. Replacing HOCl/OCl- by leucine/chloramine or alanine/chloramine in the reaction system, at pH 4.5 and 7.4, produces trace amount of 3-chlorotyrosine with the reaction yield of about 2% only. Employing the MPO-H2O2-Cl- chlorinating system at pH 5.4, production of a small amount of N-acTyr 3-chloroderivative was observed, but the reaction yield was low due to the rapid inactivation of MPO in the reaction system. The study results indicate that direct chlorination of tyrosyl residues which are not incorporated into the polypeptide structure occurs with excess HOCl/OCl- in acidic media. Due to the inability of the myeloperoxidase-H2O2-Cl- system to produce high enough HOCl concentrations, the MPO-mediated tyrosyl residue chlorination is not effective. Semistable amino-acid chloramines also appeared not effective as chlorine donors in direct tyrosyl chlorination.     

The cell death protease Kex1p is essential for hypochlorite-induced apoptosis in yeast

Following microbial pathogen invasion, the human immune system of activated phagocytes generates and releases the potent oxidant hypochlorous acid (HOCl), which contributes to the killing of menacing microorganisms. Though tightly controlled, HOCl generation by the myeloperoxidase-hydrogen peroxide-chloride system of neutrophils/monocytes may occur in excess and lead to tissue damage. It is thus of marked importance to delineate the molecular pathways underlying HOCl cytotoxicity in both microbial and human cells. Here, we show that HOCl induces the generation of reactive oxygen species (ROS), apoptotic cell death and the formation of specific HOCl-modified epitopes in the budding yeast Saccharomyces cerevisiae. Interestingly, HOCl cytotoxicity can be prevented by treatment with ROS scavengers, suggesting oxidative stress to mediate the lethal effect. The executing pathway involves the pro-apoptotic protease Kex1p, since its absence diminishes HOCl-induced production of ROS, apoptosis and protein modification. By characterizing HOCl-induced cell death in yeast and identifying a corresponding central executor, these results pave the way for the use of Saccharomyces cerevisiae in HOCl research, not least given that it combines both being a microorganism as well as a model for programmed cell death in higher eukaryotes.     

The major excreted protein (MEP) of transformed mouse cells and cathepsin L have similar protease specificity

The major excreted protein of transformed mouse cells is an acid activable cysteine protease. In this paper, oxidized insulin B chain is shown to be a substrate for this protease. By isolation and analysis of the insulin B peptides generated by the protease, the bond specificity of this protease was determined. The bonds preferentially cleaved are glu13-ala14, leu17-val18, and tyr26-thr27. No obvious preference for a specific amino acid was found in these studies. The bond specificity of this cysteine protease for oxidized insulin B chain has been compared with that of other proteases, and it is the same as that reported for cathepsin L, suggesting that the major excreted protein and cathepsin L may be the same protein.     

The Pla surface protease/adhesin of Yersinia pestis mediates bacterial invasion into human endothelial cells

Oxidation of low density lipoprotein (LDL) induced by hypochlorous acid (HOCl) leading to LDL(-), a minimally oxidized subspecies of LDL, was investigated. LDL(-) is characterized by its greater electronegativity and oxidative status, and is found in plasma in vivo. Its concentration was found to be elevated under conditions that predispose humans to atherosclerosis. We found that HOCl also converts LDL rapidly to an even more oxidized state, identified as LDL(2-), which is more electronegative than LDL(-). After milder oxidation for short durations, formation of LDL(-) takes place while less LDL(2-) is formed. Under these conditions, addition of methionine not only suppressed further oxidation of LDL but also favored the formation of LDL(-) over LDL(2-), possibly by removing chloramines at lysyl residues of LDL. The presence of lipoprotein-deficient plasma did not prevent HOCl-mediated conversion of LDL to more electronegative species. It is concluded that the HOCl-mediated conversion of LDL into more electronegative species might be physiologically relevant.     

Oxidation of flavonoids by hypochlorous acid: reaction kinetics and antioxidant activity studies

Flavonoids, plant polyphenols, ubiquitous components of human diet, are excellent antioxidants. Hypochlorous acid (HOCl), produced by activated neutrophils, is highly reactive chlorinating and oxidizing species. It has been reported earlier that flavonoids are chlorinated by HOCl. Here we show that flavonoids from flavonol subclass are also oxidized by HOCl, but only if the latter is in a large molar excess (≥?10). The kinetics of this reaction was studied by stopped-flow spectrophotometry, at different pH. We found that flavonols were oxidized by HOCl with the rate constants of the order of 10⁴–10⁵ M^?1 s^?1 at pH 7.5. Antioxidant activity of HOCl-modified flavonoids was measured by 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) method. Slightly higher antioxidant activity, compared to parent compounds, was observed for flavonols after their reaction with equimolar or moderate excess of HOCl whereas flavonols treated with high molar excess of HOCl exhibited decrease in antioxidant activity. The mechanism of flavonoid reaction with HOCl at physiological pH is proposed, and biological consequences of this reaction are discussed.     

Tellurium-based cysteine protease inhibitors: evaluation of novel organotellurium(IV) compounds as inhibitors of human cathepsin B

New organotellurium(IV) compounds with specific cysteine protease inhibitory activity were synthesized. Serine and aspartic protease activity were not affected by any of these compounds. All Te(IV) compounds tested exhibited high specific second-order constant for cathepsin B inactivation. Tellurium(IV) compound 6 was the best inhibitor of the series, showing a second-order constant of 36,000 M(-1)s(-1). This value is about 100-fold higher than the second-order rate for cysteine protease inactivation shown by the historic Te(IV) compound AS 101 (1). The inhibition was irreversible and time and concentration dependent; no saturation kinetics were observed, suggesting a direct bimolecular reaction. The results described in this paper show that the new organotellurium(IV) compounds are powerful inhibitors of cathepsin B, constituting promising potential anti-metastatic agents.     

Kinetic analysis of the role of histidine chloramines in hypochlorous acid mediated protein oxidation

Hypochlorous acid (HOCl) is a powerful oxidant generated from H(2)O(2) and chloride ions by the heme enzyme myeloperoxidase (MPO) released from activated leukocytes. In addition to its potent antibacterial effects, excessive HOCl production can lead to host tissue damage, with this implicated in human diseases such as atherosclerosis, cystic fibrosis, and arthritis. HOCl reacts rapidly with biological materials, with proteins being major targets. Chlorinated amines (chloramines) formed from Lys and His side chains and alpha-amino groups on proteins are major products of these reactions; these materials are however also oxidants and can undergo further reactions. In this study, the kinetics of reaction of His side-chain chloramines with other protein components have been investigated by UV/visible spectroscopy and stopped flow methods at pH 7.4 and 22 degrees C, using the chloramines of the model compound 4-imidazoleacetic acid and N-alpha-acetyl-histidine. The second-order rate constants decrease in a similar order (Cys > Met > disulfide bonds > Trp approximately alpha-amino > Lys > Tyr > backbone amides > Arg) to the corresponding reactions of HOCl, but are typically 5-25 times slower. These rate constants are consistent with His side-chain chloramines being important secondary oxidants in HOCl-mediated damage. These studies suggest that formation and subsequent reactions of His side-chain chloramines may be responsible for the targeted secondary modification of selected protein residues by HOCl that has previously been observed experimentally and highlight the importance of chloramine structure on their subsequent reactivity.