Reduced-oxidized difference spectral analysis and chemiluminescence-based Scatchard analysis demonstrate selective binding of myeloperoxidase to microbes.

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 µm; 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.