Rapid oxidation of dichlorodihydrofluorescin with heme and hemoproteins: formation of the fluorescein is independent of the generation of reactive oxygen species

FEBS Letters

Murakami, Akira³  Ohashi, Tomoko²  Kojo, Shosuke¹  Ishii, Tetsuro⁴  Taketani, Shigeru²  Mizutani, Atsushi² 

[1] Department of Food Science and Nutrition, Nara Women's University, Nara 630-8506, Japan;Department of Biotechnology, Kyoto Institute of Technology, Kyoto 606-8585, Japan;Department of Polymer Science and Engineering, Kyoto Institute of Technology, Kyoto 606-8585, Japan;Department of Biochemistry, Institute of Basic Medical Sciences, Tsukuba University, Tsukuba, Ibaraki 305-8575, Japan

关键词: Heme;    Reactive oxygen species;    2′;    7′-Dichlorodihydrofluorescin diacetate;    Hemoprotein;    Heme oxygenase-1;    ROS;    reactive oxygen species;    DCFH-DA;    2′;    7′-dichlorodihydrofluorescin diacetate;    DCFH;    dichlorodihydrofluorescin;    DCF;    dichlorofluorescin;    SOD;    superoxide dismutase;    HO;    heme oxygenase;    ALA;    δ-aminolevulinic acid;    PBS;    phosphate-buffered saline;    DMEM;    Dulbecco's modified Eagle's medium;    BSA;    bovine serum albumin;    FCS;    fetal calf serum;    HRP;    horseradish peroxidase;   

DOI  :  10.1016/S0014-5793(01)03262-8

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Oxidative stress and the generation of reactive oxygen species (ROS) have been implicated in the pathogenesis of cellular damage. These events have usually been reported in terms of oxidation of a reporter molecule such as 2′,7′-dichlorodihydrofluorescin diacetate (DCFH-DA). Treatment of HeLa cells with hemin or metalloporphyrins resulted in a rapid oxidation of DCFH in a time- and dose-dependent manner. This oxidation was inhibited by treatment of the cells with a large amount of superoxide dismutase and catalase, which is different from observations that these enzymes had no effect on the induction of heme oxygenase-1, a stress-induced protein, in hemin-treated cells. To examine the possibility that the oxidation of DCFH is independent of the generation of ROS, the oxidation was measured using hemoglobin-synthesizing erythroleukemia K562 cells. When K562 cells were treated with δ-aminolevulinic acid, a precursor of heme, oxidation of DCFH increased depending on the heme content in cells. Then DCFH-DA was oxidized directly with heme, hemoglobin, myoglobin and cytochrome c. These results suggest that oxidation of DCFH is not always related to the generation of ROS but may be related to heme content in cells.

【 授权许可】

Unknown