Identification of proteins containing cysteine residues that are sensitive to oxidation by hydrogen peroxide at neutral pH.
A procedure for detecting proteins that contain H(2)O(2)-sensitive cysteine (or selenocysteine) residues was developed as a means with which to study protein oxidation by H(2)O(2) in cells. The procedure is based on the facts that H(2)O(2) and biotin-conjugated iodoacetamide (BIAM) selectively and competitively react with cysteine residues that exhibit a low pK(a), and that the decrease in the labeling of cell lysate proteins with BIAM caused by prior exposure of cells to H(2)O(2) or to an agent that induces H(2)O(2) production can be monitored by streptavidin blot analysis. This procedure was applied to rat pheochromocytoma PC12 cells directly treated with H(2)O(2), mouse hippocampal HT22 cells in which H(2)O(2) production was induced by glutamate, and human erythroleukemia K562 cells in which H(2)O(2) production was induced by phorbol myristate acetate. It revealed that several cell proteins contain cysteine or selenocysteine residues that are selectively oxidized by H(2)O(2). Three of these H(2)O(2)-sensitive proteins were identified as a member of the protein disulfide isomerase family, thioredoxin reductase, and creatine kinase, all of which were previously known to contain at least one reactive cysteine or selenocysteine at their catalytic sites. This procedure should thus prove useful for the identification of proteins that are oxidized by H(2)O(2) generated in response to a variety of extracellular agents.
Kim
J R
JR
Laboratory of Cell Signaling, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Yoon
H W
HW
Kwon
K S
KS
Lee
S R
SR
Rhee
S G
SG
D000478:Alkylation; D000595:Amino Acid Sequence; D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry; D003402:Creatine Kinase / Q000032:analysis / Q000737:chemistry; D003545:Cysteine / Q000032:analysis* / Q000737:chemistry; D005029:Ethylenediamines / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D016716:PC12 Cells; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D011817:Rabbits; D051381:Rats; D019809:Streptavidin / Q000737:chemistry
D005029:Ethylenediamines; C093128:N-biotinoyl-N'-iodoacetylethylenediamine; D011506:Proteins; D001710:Biotin; D019809:Streptavidin; D006861:Hydrogen Peroxide; D003402:Creatine Kinase; D003545:Cysteine