Resonance Raman Microscopic Analysis of Hemoproteins in Living Cells

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Raman microspectroscopy is a useful method to investigate the structures of micrometer-sized particles in a non-destructive manner. In this study, we have applied Raman microspectroscopy to myeloperoxidase (MPO), a hemoprotein, in granules of neutrophils. MPO catalyzes the reaction of hydrogen peroxide and chloride ion to produce hypochlorous acid, which is highly cytotoxic and readily reacts with most biological molecules, degrading structural proteins and inactivating enzymes. The cytotoxicity of hypochlorous acid is expected to indirectly contribute to inflammatory tissue damage caused by neutrophils. As a first step of this study, we have examined the enzymatic reaction of MPO by absorption spectroscopy and found that MPO catalyzes two reactions, one to produce hypochlorous acid and the other to oxidize general substrates. In the presence of famotidine, an anti-inflammatory drug, the latter reaction in activated and the former reaction is suppressed, thus famotidine being an inhibitor of hypochlorous acid production. To reveal the binding mode of famotidine to MPO, ultraviolet resonance Raman spectra were investigated. The results have shown that famotidine binds to asite near the chloride-ion binding site and competitively inhibits the binding of chloride ion to MPO.Raman microscopic analysis of living neutrophils was conducted finally. The spectraobtained clearly show that the micro-environments of MPO in the neutrophil granules are acidic(pH 5) and the heme iron is liganded by a chloride ion, which partially inhibits the biding of the true substrate, hydrogen peroxide. This may be part of the self-defense mechanism of neurophil from the cytotoxicity of MPO.