Reduced-oxidized difference spectral analysis and chemiluminescence-based Scatchard analysis demonstrate selective binding of myeloperoxidase to microbes Robert C. Allen 1 * and Jackson T. Stephens Jr 2 ABSTRACT: Myeloperoxidase (MPO), a microbicidal haloperoxidase of neutrophil leukocytes, was observed to selectively bind to bacteria. Binding was quantified by dithionite-reduced minus oxidized (R–O) difference spectral analysis. Escherichia coli and Pseudomonas aeruginosa showed large MPO binding by R–O difference spectral analysis, whereas Streptococcus sanguinis did not. For increased sensitivity, free and microbe-bound MPO and chloroperoxidase (CPO) activities were quantified by acid-optimum haloperoxidase-dependent chemiluminescence (CL) measurements, and these data were used for Scatchard analysis. The MPO bound/free (B/F) CL ratio was 49.5 for P. aeruginosa, 14.6 for Staphylococcus aureus, 2.8 for E. coli, 0.7 for Candida albicans and 0.4 for S. sanguinis. By comparison, the CPO B/F CL ratio was 0.03 for P. aeruginosa, 0.09 for S. aureus, 0.31 for E. coli, 0.18 for C. albicans and 0.16 for S. sanguinis. As a member of the lactic acid family of bacteria and a viridans streptococcus, S. sanguinis does not synthesize cytochromes and is catalase-negative. The metabolic products of S. sanguinis, i.e. lactic acid and hydrogen peroxide, provide optimal acidity and substrate for MPO oxidation of chloride to hypochlorite. Hypochlorite can react with organic substrates to yield dehydrogenated or chlorinated products, but when peroxide is not limiting, hypochlorite reacts with peroxide yielding singlet oxygen. The reactivity of hypochlorite is dependent on substrate availability.The microsecond half-life of electronically excited singlet oxygen restricts reactivity to within a radius of <0.25 mm; i.e. the reactivity of singlet oxygen is both substrate and half-life dependent. Poor MPO binding provides protection and possibly competitive advantage to viridans streptococci. Copyright © 2010 John Wiley & Sons, Ltd. Keywords: myeloperoxidase chloroperoxidase; reduced-oxidized difference spectrum; Scatchard analysis; selective microbe binding; acid-optimum haloperoxidase chemiluminescence Introduction Healthy human adults produce about a hundred billion (~10 11 ) neutrophil leukocytes daily (1). After a circulating lifetime of about 10 h, these neutrophils leave the blood to enter the body tissues. A portion of these neutrophils migrate to the mouth, gastrointestinal tract, urinary bladder and vagina. Myeloperoxi- dase (MPO), a 145 kDa dimeric alpha-heme haloperoxidase present in azurophilic (lysosomal) granules, makes up approxi- mately 5% of the dry weight of the neutrophils (2). Assuming a neutrophil cell volume of 450 fL, a specific gravity of 1.1 and a cell water content of 84%, the rate of MPO synthesis is about 2.8 mmol (i.e. 0.4 g) of MPO per day in healthy human adults. Bone marrow synthesis of neutrophils and the MPO content per neutrophil are markedly increased in states of inflammation and with G-CSF treatment (3). MPO exerts potent and broad-spectrum killing activity against Gram-positive and Gram-negative bacteria, yeast and fungi. Although MPO can catalyze classical peroxidase activity, typically measured as dye (e.g. guaiacol) dehydrogenation to a colored product, it is the haloperoxidase activity of MPO that is respon- sible for microbe killing. Such activity requires an acid milieu, hydrogen peroxide, and an appropriate halide cofactor (4). MPO catalyzes a highly exergonic wet combustion of the microbe yielding chemiluminescence (CL) that is also pH-, halide- and peroxide-dependent (5,6). MPO binding to Gram-negative and most Gram-positive bac- teria was directly observed by the authors. However, S. sanguinis, a lactic acid bacterium (LAB), showed little or no MPO binding. Selectivity of MPO-microbe binding could influence flora compo- sition. The migration of neutrophils to mucosal spaces with pre- dominant LAB flora and acidic pH, e.g. mouth and vagina (7,8), provides a mechanism for the delivery, release, solubilization and potential binding of MPO to the microbes present. This report employs reduced-oxidized difference spectral analysis and high-sensitivity CL measurements of haloperoxidase activities to document the selective binding properties of MPO to the bacteria and yeast tested, and considers the implications of such binding selectivity. * Correspondence to: R. C. Allen, Creighton University, School of Medicine, Department of Pathology, 601 North 30th Street, Omaha, NE, 68131 USA. E-mail: robertallen@creighton.edu 1 Creighton University, School of Medicine, Department of Pathology, Omaha, NE 68131, USA 2 Exoxemis Inc., Little Rock, AR 72201, USA Research article Received: 16 November 2009, Revised: 01 February 2010, Accepted: 09 February 2009, Published online in Wiley Online Library: 30 March 2010 (wileyonlinelibrary.com) DOI 10.1002/bio.1210 208 Luminescence 2011; 26: 208–213 Copyright © 2010 John Wiley & Sons, Ltd.