NEBRASKA REDOX BIOLOGY CENTER EDUCATIONAL PORTAL

Nebraska Redox Biology Center Educational Portal


Hypochlorous Acid

Hypochlorous acid (HClO) is a weak acid formed by hydrolysis of chlorine gas. HClO is powerful oxidizer. Its stable in solution sodium hypochlorite (NaClO) or calcium hypochlorite (Ca(ClO)2) salts have good antimicrobial activity and are used as a bleach and disinfectants [ 1, 2, 3, 4 ]. Hypochlorous acid reacts with many biological molecules, especially DNA, RNA, thiols, heme proteins, amino groups, carbonhydrates and lipids [ 4, 5, 6, 7, 8, 9, 10, 11, 12 ].

Cl2 + H2O ⇄ HClO + HCl ;


Sulfur-containing compounds such as cysteine, methionine, or glutathione can be easily oxydized by Hypochlorous acid. HOCl-mediated oxidation of cysteine thiols gives rise to unstable sulfenyl chloride (R-SCl) intermediates, which react with water to form oxidized cysteine sulfenic acids (R-SOH). These highly reactive intermediates can either be reduced by members of the thioredoxin family or be further oxidized to sulfinic (R-SO2H) and sulfonic (R-SO3H) acids. Reaction of HOCl with methionine leads to methionine sulfoxide or dehydromethionine formation [ 2, 4, 6, 7, 8, 9 ].

Amines are the second most reactive targets of HOCl in proteins. Reaction with HOCl leads to formation of respective chloramines (-NHCl). Similarly to HOCl, chloramines are reactive chlorine species. They can react with iron or copper ions to generate nitrogen radicals, which are extremely reactive. HOCl reactivity resulting in amino acid modifications, including the formation of 2-oxo-histidine and 3-chlorotyrosine which known as specific biomarkers for HOCl stress in cells [ 2, 4, 6, 7, 9, 11 ].


Reactions of hypochlorous acid (HOCl) with biomolecules. Reaction of HOCl with (A) sulfur-containing compounds or (B) amines. Brackets indicate unstable reactive intermediates [ 4 ].

HOCl and chloramines can react with nucleotides and lipids. Primary and secondary amines of nucleotide bases appear to be the major targets in DNA and RNA, with the formation of chloramines preceding the formation of nitrogen radicals and stable chlorinated nucleotides. High doses of HOCl can eventually lead to DNA strand breakage. Double bonds in unsaturated fatty acids can be chlorinated to form chlorohydrins. Amines in the head group of lipids, such as those in phosphatidylethanolamine, can form chloramines, nitrogen radicals, and aldehydes. Radicals derived from HOCl reactions with amines or peroxides can lead to lipid peroxidation [ 2, 4, 5, 6, 7, 9, 10, 11, 12 ].

HOCl mediated biomolecules chlorination and oxidation [ 13, ].


Hypochlorous acid generation is one of the critical component in immune response. Activation of leukocytes is a key event that triggers the release of a range of enzymes from intracellular granules and the production of superoxide radical by NADPH oxidase complexes at the plasma membrane. Myeloperoxidase (MPO) is released extracellularly and into phagosomal compartments by neutrophils, monocytes, and some tissue macrophages, where it catalyzes the reaction of halide and pseudo-halide ions with hydrogen peroxide. The native Fe3+ heme of MPO reacts rapidly with H2O2 by a two-electron reaction to give reactive Fe(IV)-oxo porphyrin radical cation species which then catalyze the conversion of halide and pseudo-halide ions (Cl-, Br-, SCN-) to the corresponding hypohalous acids (hypochlorous acid, hypobromous acid, and hypothiocyanous acid) [ 2, 4, 14, 15, 16 ]. In addition to antimicrobial peptides (defensins) and broadly acting proteases, phagocytosis associated generation of reactive oxygen species and hypochlorous acid is regarded as the critical killing mechanism for most pathogens. HOCl is most commonly implicated as the reactive species responsible for neutrophil-mediated bacterial cell killing [ 2, 4, 14, 16, 18 ].

The MPO-H2O2-chloride antimicrobial system [ 19 ].


Major analytical methods for hypochlorous acid detection in vitro and in vivo [ 20, 21, 22, 23, 24, 25, 26 ].


1) HOCl in water can be determined by addition of bromine and fluorescein. The resulting change in pink color can be followed using spectrophotometry [ 20, 21 ].

2) Tris(2-carboxyethyl)phosphine (TCEP) oxidation method. In this metehoa the sample should be treated by TCEP and resedual ammount of TCEP is measured after reaction with 5,5'-dithiobis(2-nitrobenzoic acid) via the final reaction product, 2-nitro-5-thiobenzoate. The concentration of HOCl can calculated based on the oxidation of TCEP by HOCl in a 1:1 ratio [ 20, 21, 22 ].

3) Oxidation of fluorescein platform based probes. This highly specific and sensitive method can be used for imaging of HOCl production by living cell. Fluorescent probes have greater sensitivity, fast response time and simplicity of implementation [ 23, 24, 25 ].


Another probes for HOCl detection are based on HOCl mediated oxydation of p-methoxyphenol, thiol, alkoxyaniline, dibenzoylhydrazine, hydroxamic acid, oxim derivatives and oxazine conjugated nanoparticles [ 20, 21 ].

New highly sensitive and selective 'turn-on' fluorescent probe for HOCl detection in living cells is based on boron-dipyrromethene (BODIPY) dye, where the oxidation of thioethers into sulfoxides in the presence of HOCl is act as a sensing trigger [ 26 ].


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