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.

拉曼微光谱镜是一种有用的方法，是以非破坏性方式研究微米大小的颗粒的结构。在这项研究中，我们在中性粒细胞的颗粒中将拉曼微光谱法应用于髓过氧化物酶（MPO），一种血蛋白。 MPO催化过氧化氢和氯离子的反应产生次伐多酸，该次醛酸是高度细胞毒性的，并且很容易与大多数生物分子反应，从而降解结构蛋白和灭活酶。预期次氯酸的细胞毒性会间接导致嗜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催化了两种反应，一种反应产生次甲酸，另一个用于氧化一般的底物。在抗炎药的Famotidine存在下，激活和前反应中的后一种反应被抑制，因此Famotidine是次伐多甲酸产生的抑制剂。为了揭示Famotidine与MPO的结合模式，研究了紫外线共振拉曼光谱。结果表明，Famotidine与氯离子结合位点附近的ASITE结合，并竞争抑制氯离子与MPO的结合。最终，进行了对嗜中性粒细胞的RAMAN显微镜分析。被镜的光谱清楚地表明，嗜中性粒细胞颗粒中MPO的微环境是酸性的（pH 5），血红素铁被氯离子配体配体，氯离子会部分抑制真正的底物过氧化氢。这可能是MPO的细胞毒性的神经磷的自卫机制的一部分。