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Probing cellular protein targets of H 2 O 2 with fluorescein‐conjugated iodoacetamide and antibodies to fluorescein
Author(s) -
Wu Yalin,
Kwon Ki-Sun,
Rhee Sue Goo
Publication year - 1998
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(98)01415-x
Subject(s) - iodoacetamide , fluorescein , cysteine , intracellular , a431 cells , biochemistry , chemistry , protein tyrosine phosphatase , epidermal growth factor , tyrosine , cell surface receptor , receptor , microbiology and biotechnology , biology , cell , enzyme , fluorescence , cell cycle , physics , molecular medicine , quantum mechanics
Recent studies suggest that H 2 O 2 , 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 H 2 O 2 action have not been identified. A procedure to detect proteins with reactive cysteine residues susceptible to oxidation by intracellularly generated H 2 O 2 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 H 2 O 2 generated in response to cellular stimulation with epidermal growth factor. One of these H 2 O 2 ‐sensitive proteins was identified as protein tyrosine phosphatase 1B.