Resonance Raman Microscopic Analysis of Hemoproteins in Living Cells

拉曼显微光谱学是一种无损研究微米级粒子结构的有效方法。在这项研究中，我们将拉曼显微光谱应用于中性粒细胞颗粒中的骨髓过氧化物酶（MPO），一种血红蛋白。MPO催化过氧化氢和氯离子反应生成次氯酸，次氯酸具有很强的细胞毒性，容易与大多数生物分子发生反应，降解结构蛋白和灭活酶。次氯酸的细胞毒性可能间接导致中性粒细胞引起的炎症组织损伤。作为本研究的第一步，我们通过吸收光谱检测了MPO的酶促反应，发现MPO催化了两个反应，一个是生成次氯酸，另一个是氧化一般底物。在消炎药法莫替丁的作用下，后一种反应被激活，前一种反应被抑制，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MPO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一个位点结合，并竞争性地抑制氯离子与MPO的结合。最后对活中性粒细胞进行拉曼显微分析。获得的光谱清楚地表明，中性粒细胞颗粒中MPO的微环境是酸性的（pH为5），血红素铁被氯离子配位，这部分抑制了真正的底物过氧化氢的结合。这可能是神经细胞防御MPO细胞毒性机制的一部分。

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.