Selenium-containing amino acids are targets for myeloperoxidase-derived hypothiocyanous acid: determination of absolute rate constants and implications for biological damage

Elevated MPO (myeloperoxidase) levels are associated with multiple human inflammatory pathologies. MPO catalyses the oxidation of Cl-, Br- and SCN- by H2O2 to generate the powerful oxidants hypochlorous acid (HOCl), hypobromous acid (HOBr) and hypothiocyanous acid (HOSCN) respectively. These species are antibacterial agents, but misplaced or excessive production is implicated in tissue damage at sites of inflammation. Unlike HOCl and HOBr, which react with multiple targets, HOSCN targets cysteine residues with considerable selectivity. In the light of this reactivity, we hypothesized that Sec (selenocysteine) residues should also be rapidly oxidized by HOSCN, as selenium atoms are better nucleophiles than sulfur. Such oxidation might inactivate critical Sec-containing cellular protective enzymes such as GPx (glutathione peroxidase) and TrxR (thioredoxin reductase). Stopped-flow kinetic studies indicate that seleno-compounds react rapidly with HOSCN with rate constants, k, in the range 2.8×10(3)-5.8×10(6) M-1·s-1 (for selenomethionine and selenocystamine respectively). These values are ~6000-fold higher than the corresponding values for H2O2, and are also considerably larger than for the reaction of HOSCN with thiols (16-fold for cysteine and 80-fold for selenocystamine). Enzyme studies indicate that GPx and TrxR, but not glutathione reductase, are inactivated by HOSCN in a concentration-dependent manner; k for GPx has been determined as ~5×105 M-1·s-1. Decomposed HOSCN did not induce inactivation. These data indicate that selenocysteine residues are oxidized rapidly by HOSCN, with this resulting in the inhibition of the critical intracellular Sec-dependent protective enzymes GPx and TrxR.     

Myeloperoxidase-derived oxidants selectively disrupt the protein core of the heparan sulfate proteoglycan perlecan

The potent oxidants hypochlorous acid (HOCl) and hypobromous acid (HOBr) are produced extracellularly by myeloperoxidase, following release of this enzyme from activated leukocytes. The subendothelial extracellular matrix is a key site for deposition of myeloperoxidase and damage by myeloperoxidase-derived oxidants, with this damage implicated in the impairment of vascular cell function during acute inflammatory responses and chronic inflammatory diseases such as atherosclerosis. The heparan sulfate proteoglycan perlecan, a key component of the subendothelial extracellular matrix, regulates important cellular processes and is a potential target for HOCl and HOBr. It is shown here that perlecan binds myeloperoxidase via its heparan sulfate side chains and that this enhances oxidative damage by myeloperoxidase-derived HOCl and HOBr. This damage involved selective degradation of the perlecan protein core without detectable alteration of its heparan sulfate side chains, despite the presence of reactive GlcNH₂ residing within this glycosaminoglycan. Modification of the protein core by HOCl and HOBr (measured by loss of immunological recognition of native protein epitopes and the appearance of oxidatively-modified protein epitopes) was associated with an impairment of its ability to support endothelial cell adhesion, with this observed at a pathologically-achievable oxidant dose of 425 nmol oxidant/mg protein. In contrast, the heparan sulfate chains of HOCl/HOBr-modified perlecan retained their ability to bind FGF-2 and collagen V and were able to promote FGF-2-dependent cellular proliferation. Collectively, these data highlight the potential role of perlecan oxidation, and consequent deregulation of cell function, in vascular injuries by myeloperoxidase-derived HOCl and HOBr.     

Reactions and reactivity of myeloperoxidase-derived oxidants: differential biological effects of hypochlorous and hypothiocyanous acids

Myeloperoxidase (MPO) is recognised to play important roles both in the immune system and during the development of numerous human pathologies. MPO is released by activated neutrophils, monocytes and some tissue macrophages, where it catalyses the conversion of hydrogen peroxide to hypohalous acids (HOX; X = Cl, Br, SCN) in the presence of halide and pseudo-halide ions. The major reactive species produced by MPO under physiological conditions are hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN), with the ratio of these oxidants critically dependent on the concentration of thiocyanate ions (SCN?). The reactivity and selectivity of HOCl and HOSCN for biological targets are markedly different, indicating that SCN? ions have the potential to modulate both the extent and nature of oxidative damage in vivo. This article reviews recent developments in our understanding of the role of SCN? in modulating the formation of MPO-derived oxidants, particularly in respect to the differences in reaction kinetics and targets of HOCl compared to HOSCN and the ability of these two oxidants to induce damage in biological systems.     

Hypothiocyanite and host–microbe interactions

The pseudohypohalous acid hypothiocyanite/hypothiocyanous acid (OSCN⁻/HOSCN) has been known to play an antimicrobial role in mammalian immunity for decades. It is a potent oxidant that kills bacteria but is non-toxic to human cells. Produced from thiocyanate (SCN⁻) and hydrogen peroxide (H₂O₂) in a variety of body sites by peroxidase enzymes, HOSCN has been explored as an agent of food preservation, pathogen killing, and even improved toothpaste. However, despite the well-recognized antibacterial role HOSCN plays in host–pathogen interactions, little is known about how bacteria sense and respond to this oxidant. In this work, we will summarize what is known and unknown about HOSCN in innate immunity and recent advances in understanding the responses that both pathogenic and non-pathogenic bacteria mount against this antimicrobial agent, highlighting studies done with three model organisms, Escherichia coli, Streptococcus spp., and Pseudomonas aeruginosa.     

铜绿假单胞菌中S型绿脓杆菌素与荧光嗜铁素的功能协同性分析

在铜绿假单胞菌(Pseudomonas aeruginosa)中,S型绿脓杆菌素S2和S4与细菌中的铁载体荧光嗜铁素(pyoverdine)使用相同的摄取通道,表明二者之间存在某些联系。本研究表征了细菌中3个S型绿脓杆菌素(Pys2、PA3866、PyoS5)的单细菌基因表达分布,并研究了S2型绿脓杆菌素对细菌摄取荧光嗜铁素的影响。结果表明,在DNA损伤压力下,S型绿脓杆菌素基因的表达在细菌种群中呈现出高度分化,外源加入S2型绿脓杆菌素会减少细菌对荧光嗜铁素的摄取,因此S2型绿脓杆菌素的存在会阻止不合成荧光嗜铁素的“欺骗者”摄取环境中荧光嗜铁素,进而减弱其对活性氧(reactive oxygen species,ROS)压力的抵抗能力。另外我们发现,在细菌中过表达SOS响应(SOS response)调节因子PrtN时,荧光嗜铁素相关合成基因的表达量显著降低,进而导致荧光嗜铁素的总合成量和外分泌量显著降低。以上结果表明细菌中SOS压力响应系统与铁摄取系统的功能是存在相互联系的。     

Caffeine induces Ca2+ release by reducing the threshold for luminal Ca2+ activation of the ryanodine receptor

Myeloperoxidase, released by activated phagocytes, forms reactive oxidants by catalysing the reaction of halide and pseudo-halide ions with H(2)O(2). These oxidants have been linked to tissue damage in a range of inflammatory diseases. With physiological levels of halide and pseudo-halide ions, similar amounts of HOCl (hypochlorous acid) and HOSCN (hypothiocyanous acid) are produced by myeloperoxidase. Although the importance of HOSCN in initiating cellular damage via thiol oxidation is becoming increasingly recognized, there are limited data on the reactions of HOSCN with other targets. In the present study, the products of the reaction of HOSCN with proteins has been studied. With albumin, thiols are oxidized preferentially forming unstable sulfenyl thiocyanate derivatives, as evidenced by the reversible incorporation of (14)C from HOS(14)CN. On consumption of the HSA (human serum albumin) free thiol group, the formation of stable (14)C-containing products and oxidation of tryptophan residues are observed. Oxidation of tryptophan residues is observed on reaction of HOSCN with other proteins (including myoglobin, lysozyme and trypsin inhibitor), but not free tryptophan, or tryptophan-containing peptides. Peptide mass mapping studies with HOSCN-treated myoglobin, showed the addition of two oxygen atoms on either Trp(7) or Trp(14) with equimolar or less oxidant, and the addition of a further two oxygen atoms to the other tryptophan with higher oxidant concentrations (> or = 2-fold). Tryptophan oxidation was observed on treating myoglobin with HOSCN in the presence of glutathione and ascorbate. Thus tryptophan residues are likely to be favourable targets for the reaction in biological systems, and the oxidation products formed may be useful biomarkers of HOSCN-mediated protein oxidation.     

Discovery of an operon that participates in agmatine metabolism and regulates biofilm formation in Pseudomonas aeruginosa

Agmatine is the decarboxylation product of arginine and a number of bacteria have devoted enzymatic pathways for its metabolism. Pseudomonas aeruginosa harbours the aguBA operon that metabolizes agmatine to putrescine, which can be subsequently converted into other polyamines or shunted into the TCA cycle for energy production. We discovered an alternate agmatine operon in the P. aeruginosa strain PA14 named agu2ABCA′ that contains two genes for agmatine deiminases (agu2A and agu2A′). This operon was found to be present in 25% of clinical P. aeruginosa isolates. Agu2A′ contains a twin‐arginine translocation signal at its N‐terminus and site‐directed mutagenesis and cell fractionation experiments confirmed this protein is secreted to the periplasm. Analysis of the agu2ABCA′ promoter demonstrates that agmatine induces expression of the operon during the stationary phase of growth and during biofilm growth and agu2ABCA′ provides only weak complementation of aguBA, which is induced during log phase. Biofilm assays of mutants of all three agmatine deiminase genes in PA14 revealed that deletion of agu2ABCA′, specifically its secreted product Agu2A′, reduces biofilm production of PA14 following addition of exogenous agmatine. Together, these findings reveal a novel role for the agu2ABCA′ operon in the biofilm development of P. aeruginosa.     

Myeloperoxidase-derived oxidants inhibit sarco/endoplasmic reticulum Ca2+-ATPase activity and perturb Ca2+ homeostasis in human coronary artery endothelial cells

The sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) plays a critical role in Ca(2+) homeostasis via sequestration of this ion in the sarco/endoplasmic reticulum. The activity of this pump is inhibited by oxidants and impaired in aging tissues and cardiovascular disease. We have shown previously that the myeloperoxidase (MPO)-derived oxidants HOCl and HOSCN target thiols and mediate cellular dysfunction. As SERCA contains Cys residues critical to ATPase activity, we hypothesized that HOCl and HOSCN might inhibit SERCA activity, via thiol oxidation, and increase cytosolic Ca(2+) levels in human coronary artery endothelial cells (HCAEC). Exposure of sarcoplasmic reticulum vesicles to preformed or enzymatically generated HOCl and HOSCN resulted in a concentration-dependent decrease in ATPase activity; this was also inhibited by the SERCA inhibitor thapsigargin. Decomposed HOSCN and incomplete MPO enzyme systems did not decrease activity. Loss of ATPase activity occurred concurrent with oxidation of SERCA Cys residues and protein modification. Exposure of HCAEC, with or without external Ca(2+), to HOSCN or HOCl resulted in a time- and concentration-dependent increase in intracellular Ca(2+) under conditions that did not result in immediate loss of cell viability. Thapsigargin, but not inhibitors of plasma membrane or mitochondrial Ca(2+) pumps/channels, completely attenuated the increase in intracellular Ca(2+) consistent with a critical role for SERCA in maintaining endothelial cell Ca(2+) homeostasis. Angiotensin II pretreatment potentiated the effect of HOSCN at low concentrations. MPO-mediated modulation of intracellular Ca(2+) levels may exacerbate endothelial dysfunction, a key early event in atherosclerosis, and be more marked in smokers because of their higher SCN(-) levels.     

Hypochlorite- and hypobromite-mediated radical formation and its role in cell lysis.

Activated leukocytes generate the potent oxidants HOCl and HOBr via the formation of H(2)O(2) and the release of peroxidase enzymes (myeloperoxidase, eosinophil peroxidase). HOCl and HOBr are potent microbiocidal agents, but excessive or misplaced production can cause tissue damage and cell lysis. In this study it is shown that HOBr induces red blood cell lysis at approximately 10-fold lower concentrations than HOCl, whereas with monocyte (THP1) and macrophage (J774) cells HOCl and HOBr induce lysis at similar concentrations. The role of radical formation during lysis has been investigated by EPR spin trapping, and it is shown that reaction of both oxidants with each cell type generates cell-derived radicals. Red blood cells exposed to nonlytic doses of HOCl generate novel nitrogen-centered radicals whose formation is GSH dependent. In contrast, HOBr gives rise to nitrogen-centered, membrane-derived protein radicals. With lytic doses of either oxidant, protein (probably hemoglobin)-derived, nitrogen-centered radicals are observed. Unlike the red blood cells, treatment of monocytes and macrophages with HOCl gives significant radical formation only under conditions where cell lysis occurs concurrently. These radicals are nitrogen-centered, cell-protein-derived species and have parameters identical to those detected with red blood cells and HOBr. Exposure of these cells to HOBr did not give detectable radicals. Overall these experiments demonstrate that HOCl and HOBr react with different selectivity with cellular targets, and that this can result in radical formation. This radical generation can precede, and may play a role in, cell lysis.     

Inactivation of thiol-dependent enzymes by hypothiocyanous acid: role of sulfenyl thiocyanate and sulfenic acid intermediates

Myeloperoxidase (MPO) forms reactive oxidants including hypochlorous and hypothiocyanous acids (HOCl and HOSCN) under inflammatory conditions. HOCl causes extensive tissue damage and plays a role in the progression of many inflammatory-based diseases. Although HOSCN is a major MPO oxidant, particularly in smokers, who have elevated plasma thiocyanate, the role of this oxidant in disease is poorly characterized. HOSCN induces cellular damage by targeting thiols. However, the specific targets and mechanisms involved in this process are not well defined. We show that exposure of macrophages to HOSCN results in the inactivation of intracellular enzymes, including creatine kinase (CK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In each case, the active-site thiol residue is particularly sensitive to oxidation, with evidence for reversible inactivation and the formation of sulfenyl thiocyanate and sulfenic acid intermediates, on treatment with HOSCN (less than fivefold molar excess). Experiments with DAz-2, a cell-permeable chemical trap for sulfenic acids, demonstrate that these intermediates are formed on many cellular proteins, including GAPDH and CK, in macrophages exposed to HOSCN. This is the first direct evidence for the formation of protein sulfenic acids in HOSCN-treated cells and highlights the potential of this oxidant to perturb redox signaling processes.     

Impact of myeloperoxidase-derived oxidants on the product profile of human 5-lipoxygenase

Human 5-lipoxygenase (5-LOX) oxidizes arachidonic acid to 5S-hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-HpETE) and leukotriene (LT) A₄. In neutrophils, LTA₄ is further converted to the potent chemoattractant LTB₄. These cells also contain the heme enzyme myeloperoxidase (MPO), which produces several potent oxidants such as hypochlorous acid (HOCl), which are involved in pathogen defense and immune regulation. Here, we addressed the question whether MPO-derived oxidants are able to affect the activity of 5-LOX and the product profile of this enzyme. Human 5-LOX was incubated with increasing amounts of HOCl or HOBr. Afterward, arachidonic acid metabolites of 5-LOX were analyzed by reverse-phase high-performance liquid chromatography as well as by liquid chromatography–electrospray ionization–tandem mass spectrometry. The incubation of 5-LOX with the MPO-derived oxidants significantly changed the product profile of 5-LOX. Thereby, HOCl and HOBr increased the ratio of 5-H(p)ETE to 6-trans-LTB₄ in a concentration-dependent manner. At low oxidant concentrations, there was a strong decrease in the yield of 6-trans-LTB₄, whereas 5-HpETE did not change or increased. Additionally, the formation of 8-HpETE and 12-HpETE by 5-LOX rose slightly with increasing HOCl and HOBr. Comparable results were obtained with the MPO–H₂O₂–Cl⁻ system when glucose oxidase and glucose were applied as a source of H₂O₂. This was necessary because of a strong impairment of 5-LOX activity by H₂O₂. In summary, MPO-derived oxidants showed a considerable impact on 5-LOX, impairing the epoxidation of 5-HpETE, whereas the hydroperoxidation of arachidonic acid was unaffected. Apparently, this was caused by an oxidative modification of critical amino acid residues of 5-LOX. Further work is necessary to assess the specific type and position of oxidation in the substrate-binding cavity of 5-LOX and to specify whether this interaction between 5-LOX and MPO-derived oxidants also takes place in stimulated neutrophils.     

Selective Metabolism of Hypothiocyanous Acid by Mammalian Thioredoxin Reductase Promotes Lung Innate Immunity and Antioxidant Defense

The endogenously produced oxidant hypothiocyanous acid (HOSCN) inhibits and kills pathogens but paradoxically is well tolerated by mammalian host tissue. Mammalian high molecular weight thioredoxin reductase (H-TrxR) is evolutionarily divergent from bacterial low molecular weight thioredoxin reductase (L-TrxR). Notably, mammalian H-TrxR contains a selenocysteine (Sec) and has wider substrate reactivity than L-TrxR. Recombinant rat cytosolic H-TrxR1, mouse mitochondrial H-TrxR2, and a purified mixture of both from rat selectively turned over HOSCN (k_cat = 357 ± 16 min⁻¹; K_m = 31.9 ± 10.3 μm) but were inactive against the related oxidant hypochlorous acid. Replacing Sec with Cys or deleting the final eight C-terminal peptides decreased affinity and turnover of HOSCN by H-TrxR. Similarly, glutathione reductase (an H-TrxR homologue lacking Sec) was less effective at HOSCN turnover. In contrast to H-TrxR and glutathione reductase, recombinant Escherichia coli L-TrxR was potently inhibited by HOSCN (IC₅₀ = 2.75 μm). Similarly, human bronchial epithelial cell (16HBE) lysates metabolized HOSCN, but E. coli and Pseudomonas aeruginosa lysates had little or no activity. HOSCN selectively produced toxicity in bacteria, whereas hypochlorous acid was nonselectively toxic to both bacteria and 16HBE. Treatment with the H-TrxR inhibitor auranofin inhibited HOSCN metabolism in 16HBE lysates and significantly increased HOSCN-mediated cytotoxicity. These findings demonstrate both the metabolism of HOSCN by mammalian H-TrxR resulting in resistance to HOSCN in mammalian cells and the potent inhibition of bacterial L-TrxR resulting in cytotoxicity in bacteria. These data support a novel selective mechanism of host defense in mammals wherein HOSCN formation simultaneously inhibits pathogens while sparing host tissue.     

Green fluorescent protein-expressing Escherichia coli as a selective probe for HOCl generation within neutrophils

Escherichia coli were transformed by electroporation to introduce a plasmid harboring a GFP gene-containing vector. The fluorescence of the purified GFP isolated from the transformant was quenched by myeloperoxidase (MPO)-generated HOCl, by peroxynitrous acid (ONOOH) and by enzymatically or radiolytically generated NO(2)(.) but not by other putative neutrophil-generated oxidants. Fluorescence from the bacterium was effectively quenched by HOCl but not peroxynitrite, oxidizing radicals derived from its O-O bond homolysis, or the other oxidants under study. Exposure of serum-opsonized bacteria to human neutrophils resulted in extensive loss of GFP fluorescence; fluorescence microscopy revealed that phagocytosed bacteria were completely quenched but that bacteria remaining in the external media were unquenched. Addition of sodium azide to the medium to inhibit MPO prevented neutrophil-mediated fluorescence quenching. Because the amount of HOCl required to inhibit bacterial fluorescence was an order of magnitude greater than required to inhibit colonial growth, these results imply that sufficient HOCl was formed within the neutrophil phagosome to kill the microbe.     

Light emission from tryptophan oxidation by hypobromous acid

The emission of ultraweak light from cells is a phenomenon associated with the oxidation of biomolecules by reactive oxygen species. The indole moiety present in tryptophan, serotonin and melatonin is frequently associated with the emission of light during the oxidation of these metabolites. This study presents results for hypobromous acid (HOBr) oxidation of tryptophan as a putative endogenous source of ultraweak light emission. We found that chemiluminescence elicited by the oxidation of tryptophan by HOBr was significantly higher than by hypochlorous acid (HOCl). This difference was related to secondary oxidation reactions, which were more intense using HOBr. The products identified during oxidation by HOCl, but depleted by using HOBr, were N‐formylkynurenine, kynurenine, 1,2,3,3a,8,8a‐hexahydro‐3a‐hydroxypyrrolo[2,3‐b]‐indole‐2‐carboxylic acid, oxindolylalanine and dioxindolylalanine. The emission of light is dependent on the free α‐amino group of tryptophan, and hence, the indole of serotonin and melatonin, although efficiently oxidized, did not produce chemiluminescence. The emission of light was even greater using taurine monobromamine and dibromamine as the oxidant compared to HOBr. A mechanism based on bromine radical intermediates is suggested for the higher efficiency in light emission. Altogether, the experimental evidence described in the present study indicates that the oxidation of free tryptophan or tryptophan residues in proteins is an important source of ultraweak cellular emission of light. This light emission is increased in the presence of taurine, an amino acid present in large amounts in leukocytes, where this putative source of ultraweak light emission is even more relevant. Copyright © 2012 John Wiley & Sons, Ltd.     

Thiocyanate supplementation decreases atherosclerotic plaque in mice expressing human myeloperoxidase

Abstract

Elevated levels of the heme enzyme myeloperoxidase (MPO) are associated with adverse cardiovascular outcomes. MPO predominantly catalyzes formation of the oxidants hypochlorous acid (HOCl) from Cl^?, and hypothiocyanous acid (HOSCN) from SCN^?, with these anions acting as competitive substrates. HOSCN is a less powerful and more specific oxidant than HOCl, and selectively targets thiols; such damage is largely reversible, unlike much HOCl-induced damage. We hypothesized that increased plasma SCN^?, and hence HOSCN formation instead of HOCl, may decrease artery wall damage. This was examined using high-fat fed atherosclerosis-prone LDLR^–/– mice transgenic for human MPO, with and without SCN^? (10 mM) added to drinking water. Serum samples, collected fortnightly, were analyzed for cholesterol, triglycerides, thiols, MPO, and SCN^?; study-long exposure was calculated by area under the curve (AUC). Mean serum SCN^? concentrations were elevated in the supplemented mice (200–320 μM) relative to controls (< 120 μM). Normalized aortic root plaque areas at sacrifice were 26% lower in the SCN^?-supplemented mice compared with controls (P = 0.0417), but plaque morphology was not appreciably altered. Serum MPO levels steadily increased in mice on the high-fat diet, however, comparison of SCN^?-supplemented versus control mice showed no significant changes in MPO protein, cholesterol, or triglyceride levels; thiol levels were decreased in supplemented mice at one time-point. Plaque areas increased with higher cholesterol AUC (r = 0.4742; P = 0.0468), and decreased with increasing SCN^? AUC (r = ? 0.5693; P = 0.0134). These data suggest that increased serum SCN^? levels, which can be achieved in humans by dietary manipulation, may decrease atherosclerosis burden.     

Detection of the halogenating activity of heme peroxidases in leukocytes by aminophenyl fluorescein

Abstract

The formation of hypochlorous and hypobromous acids by heme peroxidases is a key property of certain immune cells. These products are not only involved in defense against pathogenic microorganisms and in regulation of inflammatory processes, but contribute also to tissue damage in certain pathologies. After a short introduction about experimental approaches for the assessment of the halogenating activity in vitro and in cell suspensions, we are focusing on novel applications of fluorescent dye systems to detect the formation of hypochlorous acid (HOCl) in leukocytes. Special attention is directed to properties and applications of the non-fluorescent dye aminophenyl fluorescein that is converted by HOCl, HOBr, and other strong oxidants to fluorescein. This dye allows the detection of the halogenating activity in samples containing free myeloperoxidase and eosinophil peroxidase as well as in intact granulocytes using fluorescence spectroscopy and flow cytometry, respectively.     

Enzyme-based formulations for decontamination: current state and perspectives

Development of noncorrosive, cost-effective, environmentally benign, and broad-spectrum antimicrobial formulations is necessary for clinical, industrial, and domestic purposes. Many current decontaminating formulations are effective, but they require the use of strong oxidizing agents or organic solvents that have deleterious effects on human health and the surrounding environment. The emergence of antibiotic-resistant pathogens has motivated researchers to develop enzyme-based self-decontaminating formulations as alternatives to such chemical decontamination approaches. Hydrolytic and oxidative enzymes can be used to deactivate pathogens, including bacteria, spores, viruses, and fungi. Laccases, haloperoxidases, and perhydrolases catalyze the generation of biocidal oxidants, such as iodine, bromine, hypohalous acid (e.g., HOCl or HOBr), and peracetic acid. These oxidants have broad-spectrum antimicrobial activity. Due to the multi-pathway action of these oxidants, it has proven extremely difficult for microbes to gain resistance. Thus far, few examples have been reported on enzyme-based antimicrobial formulations. For these reasons, various enzyme-containing antimicrobial formulations are highlighted in this review.