Proc. Nati Acad. Sci. USA Vol. 78, No. 1, pp. 210-214, January 1981 Biochemistry Biological reactivity ofhypochlorous acid: Implications for microbicidal mechanisms ofleukocyte myeloperoxidase (bactericide/biological oxidation/neutrophil/respiration/toxin) J. MICHAEL ALBRICH, CAROL A. MCCARTHY, AND JAMES K. HURST* Department ofChemistry and Biochemical Sciences, Oregon Graduate Center, Portland, Oregon 97006 Communicated by Henry Taube, October 6, 1980 ABSTRACT Oxidative degradation ofbiological substrates by electrophile-nucleophile interactions involving association of hypochlorous acid has been examined under reaction conditions electropositive chlorine with electron-rich centers on the sub- similar to those found in active phagosomes. Iron sulfur proteins are bleached extremely rapidly, followed in decreasing order by strate; reaction pathways are-correspondingly highly dependent f3carotene, nucleotides, porphyrins, and heme proteins. En- upon medium-conditions, particularly H' and Cl- concentra- zymes containing essential cysteine molecules are inactivated with tions (14). With the exception ofamines and amino acids (11, 12, an effectiveness that roughly parallels the nucleophilic reactivities 15), the reactions of biological compounds with HOC1 are not of their sulfhydryl groups. Other compounds, including glucos- understood (16). amines, qumiones, riboflavin, and, except for N-chlorination, We report 'here the results of a survey of simple biological phospholipids, are unreactive. Rapid irreversible oxidation ofcy- compounds which can be taken as prototypic ofvarious tochromes, adenine nucleotides, and carotene pigments occurs compo- when bacterial cells are exposed to exogenous hypochlorous acid; nents of bacterial cells.:The data demonstrate that HOCI is with Escherichia coli, titrimetric oxidation of cytochrome was strongly selective -toward nucleotides and compounds that are found to coincide with loss ofaerobic respiration. The occurrence models for certain components ofrespiratory redox chains. This of these cellular reactions implicates hypochlorous acid as a pri- selectivity is shownito extend to bacterial cells. mary microbicide in myelo.roxidase-containing leukocytes; the reactivity patterns observed are consistent with the view that bac- MATERIALS AND METHODS tericidal action results primarily from loss ofenergy-linked respi- ration due to destruction ofcellular electron transport chains and Reagents. HOCI was prepared by vacuum distilling Chlorox the adenine nucleotide pool. (NaOCl) after adjusting the pH to 7-8 with dilute sulfuric acid; concentrations were determined by spectrophotometric [E2W Bacteria commonly lose their ability to divide within minutes of = 100 (17)] or iodometric'analyses. Ethanolchloramine was pre- encountering phagocytosing leukocytes (1). Loss ofcell viability pared by reaction ofHOC1 with a 3-fold excess ofethanolamine often occurs well before the onset ofcellular digestion as deter- in 0.1 M phosphate buffer at pH 5.0. Aldolase (EC 4.1.2.13) was mined by physiological changes (2), macromolecular degrada- prepared from brewer's yeast (Standard Brands) by ammonium tion (3, 4), or loss of macromolecular biosynthesis (5). The spe- sulfate precipitation (18) in the presence of proteolysis inhibi- cific reactions giving rise to cellular death have not yet been tors (19); the purified enzyme had a specific activity of 64 units/ identified, at least in part because leukocytes are capable ofini- mg of protein. Other enzymes and reagent-grade biological tiating a diverse set of processes which are potentially lethal (6, compounds were used as obtained from commercial suppliers. 7). Given the above observations, however, the microbicidal re- Escherichia coli (ATCC 25922) were grown on media 2 (20) plus actions must be among the first that attend interaction with the succinate (2 g/liter) at pH 6.5 and harvested in stationary phase. leukocyte. The bacteria were centrifuged, washed, and resuspended in Reactions catalyzed by myeloperoxidase (MPOase) appear buffer immediately before use. Sarcina lutea (Mwrococcus lu- to make major contributions to the microbicidal-action of poly- teus, generously provided by S. J. Klebanoff, University of morphonuclear leukocytes (PMNs) (6, 7). The cell-free Washington Medical School, Seattle) were grown on 5% tryptic MPOase-H202-Cl- system is 'potentially microbicidal; chlori- soy agar plates (Prepared Media Laboratories); suspensions nation of bacteria by the cell-free system (8, 9) and of macro- were made by carefully scraping the cells from the plates and molecular fractions during PMN digestion of bacteria (9) sug- agitating them in buffer. Bacterial cell concentrations were de- gests that the toxic mechanisms may involve,direct reaction with termined by phase-contrast microscopy with a hemocytometer. hypochlorous acid (HOCI), the oxidized product of MPOase HOCI Oxidations. Reaction with biological compounds was catalysis. Because HOCI is freely diffusible from the enzyme monitored byusingeither a Cary 16 spectrophotometer or Gib- (10) and the gross features of cellular reactions with phagocy- son-type stopped-flow apparatus (21). Rate laws were deter- tosing PMNs, the MPOase-H202Cl system, and exogenous mined from initial rates ofbleaching; rate constants for the com- HOC1 are similar (4, 8, 11-13), it is likely that the actual mi- pounds listed in Table 1 were -determined from loss of their crobicidal reactions are uncatalyzed. If so, minimal require- characteristic absorption bands when HOC1 was in large excess. ments for toxicity would seem to be that HOCl show high se- The reactions are generally complex, involving numerous oxi- lectivity in its reactions with biological substrates and that its dized intermediates. Any rapid initial degradative steps that did action be directed against susceptible sites-e.g., specific en- not give rise to large spectral changes (e.g., heterocyclic ring ergy-generating or biosynthetic systems. chlorination -would go undetected. Consequently, the rate con- In general, rates 'of oxidation and chlorination by various stants reported would be lower limits for these steps. Enzyme monovalent chlorine compounds can be understood in terms of inactivation was studied by addingportions ofHOC1 to solutions The publication costs ofthis article were defrayed in part by page charge Abbreviations: MPOase, myeloperoxidase; PMN; polymorphonuclear payment. This article must therefore be hereby marked "advertise- leukocyte. ment" in accordance with 18 U. S. C. §1734 solely to-indicate this fact. * To whom reprint requests should be addressed. 210 Downloaded by guest on September 28, 2021 Biochemistry: Albrich et aL Proc. NatLAcad. Sci. USA 78 (1981) 211 containing freshly dialyzed enzyme and then measuring, by ap- =12:1. Immediate reaction products had masses that increased propriate standard assay procedures, the residual activity ofali- in units of 32 ± 1 atomic mass units, suggesting epoxidation quots taken from the reaction solution. In the enzyme and cel- through intermediary formation of chlorohydrins. Because re- lular oxidation studies, HOCI was added as a bolus to rapidly tinol oxidation by HOCl gives products with masses increasing stirred reactant solutions. HOCI-promoted loss ofbacterial res- by 16 ± 1 atomic mass units, the opposite ends of the carotene piration was measured with a Clark electrode and recorder as- molecule may be reacting largely independently. /-Carotene is sembly (14). also bleached by the MPO-H202-Cl- system (24). Optical difference spectra of bacterial suspensions were re- Oxidation of numerous porphyrins, hemes, and hemopro- corded on an Aminco DW-2 spectrophotometer. Cellular nu- teins, including protoporphyrin IX, mesoporphyrin IX di- cleotide concentrations were determined, by using an Aminco methyl ester, 2-formyl4-vinyl-deuteroporphyrin dimethyl es- Chem-Glow photometer, from the integrated light intensity of ter, 2,4-diformyldeuteroporphyrin dimethyl ester, the luciferin-luciferase assay (22) taken over the first 3 sec ofre- ferriprotoporphyrin IX (hemin) and its dimethyl ester, ferridi- action. In the nucleotide studies, reactions after exposure to acetyldeuteroporphyrin, cobalt(III) mesoporphyrin IX di- HOCl were quenched by rapidly withdrawing samples into a methyl ester, myoglobin, microperoxidase, and cytochrome c, spring-loaded syringe containing perchloric acid. Intervals be- also occurred rapidly. Porphyrin and heme solutions contained tween mixing and quenching (10-300 sec) were chosen so that 1% NaDodSO4 to solubilize the macrocycle. Cytochrome c oxi- reaction with HOCI was completed and subsequent intracellu- dation by the cell-free MPO-H202-Cl- system is also rapid (un- lar reactions were minimized. In several experiments, the re- published data). action was quenched with sodium thiosulfate before acidifica- Of the porphyrin compounds examined in detail (protopor- tion to ensure that oxidation was not due to molecular chlorine phyrin IX, mesoporphyrin IX dimethyl ester, hemin, and cyto- formed from residual HOCL. The results were identical to those chrome c), all except protoporphyrin IX exhibited bleaching for runs in which thiosulfate quenching was omitted. Mass spec- rates that increased with increasing H' concentration in the pH tra were recorded on a Hitachi-Perkin-Elmer RMU-7 instru- region 4-7, although the dependency was complex and less than ment operated in the field desorption mode. first-order; protoporphyrin IX rates were inversely dependent upon acidity in this region.