Resonance Raman Microscopic Analysis of Hemoproteins in Living Cells

拉曼显微光谱是一种非破坏性地研究微米级颗粒结构的有用方法。在这项研究中，我们已经应用拉曼显微光谱髓过氧化物酶（MPO），血红素蛋白，在颗粒的中性粒细胞。MPO催化过氧化氢和氯离子反应产生次氯酸，次氯酸具有高度细胞毒性，易于与大多数生物分子反应，降解结构蛋白并使酶失活。预期次氯酸的细胞毒性会间接导致中性粒细胞引起的炎性组织损伤。作为本研究的第一步，我们已经研究了MPO的酶促反应的吸收光谱，发现MPO催化两个反应，一个产生次氯酸和其他氧化一般底物。在抗炎药法莫替丁的存在下，后一反应被激活，前一反应被抑制，因此法莫替丁是次氯酸产生的抑制剂。为了揭示法莫替丁与髓过氧化物酶的结合模式，研究了紫外共振拉曼光谱。结果表明，法莫替丁与氯离子结合位点附近的asite结合，竞争性抑制氯离子与MPO的结合。最后，对活中性粒细胞进行了拉曼显微分析。所得光谱清楚地表明，MPO在中性粒细胞颗粒中的微环境是酸性的（pH 5），并且血红素铁被氯离子配位，这部分地抑制了真正的底物过氧化氢的结合。这可能是嗜神经细胞对MPO细胞毒作用的自我防御机制的一部分。

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.