Reversible oxidation of the active site cysteine of peroxiredoxins to cysteine sulfinic acid. Immunoblot detection with antibodies specific for the hyperoxidized cysteine-containing sequence.
We previously suggested that oxidation of the active site cysteine of peroxiredoxin (Prx) I or Prx II to cysteine sulfinic acid in H2O2-treated cells is reversible (Woo, H. A., Chae, H. Z., Hwang, S. C., Yang, K.-S., Kang, S. W., Kim, K., and Rhee, S. G. (2003) Science 300, 653-656). In contrast, it was recently proposed that sulfinylation of Prx II, but not that of Prx I or Prx III, is reversible (Chevallet, M., Wagner, E., Luche, S., van Dorssealaer, A., Leize-Wagner, E., and Rabilloud, T. (2003) J. Biol. Chem. 278, 37146-37153). The detection of sulfinylated proteins in both of these previous studies relied on complex proteomics analysis. We now describe a simple immunoblot assay for the detection of sulfinylated Prx enzymes that is based on antibodies produced in response to a sulfonylated peptide modeled on the conserved active site sequence. These antibodies recognized both sulfinic and sulfonic forms of Prx equally well and allowed the detection of sulfinylated Prx enzymes in H2O2-treated cells with high sensitivity and specificity. With the use of these antibodies, we demonstrated that not only the cytosolic enzymes Prx I and Prx II but also the mitochondrial enzyme Prx III undergo reversible sulfinylation. The generation of antibodies specific for sulfonylated peptides should provide insight into protein function similar to that achieved with antibodies to peptides containing phosphoserine or phosphothreonine.
Woo
Hyun Ae
HA
Center for Cell Signaling Research and Division of Molecular Life Sciences, Ewha Womans University, Seoul 120-750, Korea.
Kang
Sang Won
SW
Kim
Hyung Ki
HK
Yang
Kap-Seok
KS
Chae
Ho Zoon
HZ
Rhee
Sue Goo
SG
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D015151:Immunoblotting / Q000379:methods*; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D010544:Peroxidases / Q000737:chemistry*; D054464:Peroxiredoxins; D010768:Phosphoserine / Q000737:chemistry; D010769:Phosphothreonine / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D012680:Sensitivity and Specificity; D013441:Sulfinic Acids / Q000737:chemistry*; D013997:Time Factors
D010455:Peptides; D011506:Proteins; D013441:Sulfinic Acids; D010769:Phosphothreonine; D010768:Phosphoserine; D006861:Hydrogen Peroxide; D010544:Peroxidases; D054464:Peroxiredoxins; D003545:Cysteine; D010100:Oxygen