Resonance Raman Microscopic Analysis of Hemoproteins in Living Cells

活细胞中血红素蛋白的共振拉曼显微镜分析

基本信息

批准号：
09680643
负责人：
TAKEUCHI Hideo
金额：
$ 1.73万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

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.

拉曼显微光谱是一种以非破坏性方式研究微米级颗粒结构的有用方法。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髓过氧化物酶（MPO）（一种血红素蛋白）。 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，容易与大多数生物分子发生反应，降解结构蛋白并使酶失活。次氯酸的细胞毒性预计会间接导致中性粒细胞引起的炎症组织损伤。作为这项研究的第一步，我们通过吸收光谱检查了 MPO 的酶促反应，发现 MPO 催化两个反应，一个产生次氯酸，另一个氧化一般底物。在抗炎药法莫替丁存在下，后一个反应被激活，前一个反应被抑制，因此法莫替丁是次氯酸生成的抑制剂。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位点结合，竞争性抑制氯离子与MPO的结合。最后对活体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 5），并且血红素铁由氯离子配体，这部分抑制了真正的底物过氧化氢的结合。这可能是神经细胞免受 MPO 细胞毒性的自我防御机制的一部分。