? While reactive oxygen species, including H2O2, have been shown to modulate a number of kinases, phosphatases, redox-sensitive transcription factors, and genes involved in cellular events including apoptosis and cancer, little information is available as to the proteins directly targeted by H2O2. The primary target of H2O2-linked protein modification is likely to be cysteine residues that are converted first to cysteine sulfenic acids (Cys-SOH), then either directly re-reduced or further modified to form disulfide bonds or other oxidized species. Due to the unstable nature of the sulfenic acid intermediate, identification of proteins containing this species has been difficult. To provide a tool to identify and monitor redox sensitive proteins, an assay will be developed and validated to monitor Cys-SOH appearance and disappearance in vivo after stimulation with reactive oxygen species. With this method in hand, we will be able to identify which proteins are modified, to determine the reactive cysteines in each of these proteins, and to determine if different stimulants, including a representative group of peptide growth factors, cytokines, and peroxides, mediate Cys-SOH formation on different proteins. Finally, the last specific aim will focus on how Cys-SOH formation could be specific to a limited number of proteins by investigating whether or not a low pKa value for the reactive cysteine is critical to the formation of this intermediate. ? ?
