Probing cellular protein targets of H2O2 with fluorescein‐conjugated iodoacetamide and antibodies to fluorescein

FEBS Letters

Kwon, Ki-Sun¹  Rhee, Sue Goo¹  Wu, Yalin¹ 

[1] Laboratory of Cell Signaling, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 3, Room 122, Bethesda, MD 20892, USA

关键词: Hydrogen peroxide;    Intracellular messenger;    Cysteine oxidation;    Growth factor;    Protein tyrosine phosphatase;    EGF;    epidermal growth factor;    PTP1B;    protein tyrosine phosphatase 1B;    5-IAF;    5-iodoacetamidofluorescein;    DPI;    diphenyleneiodonium iodide;    PAGE;    polyacrylamide gel electrophoresis;    ECL;    enhanced chemiluminescence;    BSA;    bovine serum albumin;   

DOI  :  10.1016/S0014-5793(98)01415-X

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Recent studies suggest that H₂O₂, at subtoxic concentrations generated in response to the activation of a variety of cell surface receptors, functions as an intracellular messenger. However, the intracellular targets of H₂O₂ action have not been identified. A procedure to detect proteins with reactive cysteine residues susceptible to oxidation by intracellularly generated H₂O₂ is now described. This approach is based on the labeling of proteinaceous cysteine with 5-iodoacetamidofluorescein at pH 5.5 and immunoblot analysis of the labeled proteins with antibodies specific to fluorescein. With this procedure, many proteins in human A431 cells were shown to contain reactive cysteines and to be readily oxidized by H₂O₂ generated in response to cellular stimulation with epidermal growth factor. One of these H₂O₂-sensitive proteins was identified as protein tyrosine phosphatase 1B.

【 授权许可】

Unknown