Real-time assays for monitoring the influence of sulfide and sulfane sulfur species on protein thiol redox states.
Hydrogen sulfide (H2S) is known to induce persulfidation of protein thiols. However, the process of H2S-induced persulfidation is not fully understood as it requires an additional oxidant. There are several mechanistic possibilities and it is of interest to determine which pathway is kinetically most relevant. Here, we detail in vitro assays for the real-time monitoring of thiol redox states in two model proteins with oxidizable cysteines, PTEN, and roGFP2. These allow kinetic measurements of the response of defined protein thiols (or disulfides) to sulfide and sulfane sulfur species. The combination of these assays with cold cyanolysis reveals the role of intermediary sulfane sulfur species in H2S-induced protein thiol oxidation.
Division of Redox Regulation, German Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance, Heidelberg, Germany.
Romy
R
Greiner
Division of Redox Regulation, German Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance, Heidelberg, Germany. Electronic address: t.dick@dkfz.de.
Tobias P
TP
Dick
D003545:Cysteine / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000737:chemistry; D006801:Humans; D006862:Hydrogen Sulfide / Q000737:chemistry*; D006923:Hymecromone / Q000031:analogs & derivatives / Q000737:chemistry; D007700:Kinetics; D010084:Oxidation-Reduction; D051059:PTEN Phosphohydrolase / Q000032:analysis* / Q000737:chemistry; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D015398:Signal Transduction; D013440:Sulfides / Q000737:chemistry; D013861:Thiocyanates / Q000737:chemistry
C119285:6,8-difluoro-4-methylumbelliferyl phosphate; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D013440:Sulfides; D013861:Thiocyanates; C051552:persulfides; D049452:Green Fluorescent Proteins; D006923:Hymecromone; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human; D003545:Cysteine; C031760:thiocyanate; C033479:sodium sulfide; D006862:Hydrogen Sulfide
Cyanolysis; Hydrogen sulfide; PTEN; Persulfidation; Polysulfides; Protein thiols; Sulfane sulfur; Sulfhydration; roGFP2