Decreased S-nitrosation of peptide thiols in the membrane interior

It has been proposed that autoxidation of nitric oxide (NO) stimulates S-nitrosation of thiols located in the hydrophobic milieu. We tested whether thiols located in hydrophobic membranes undergo enhanced S-nitrosation in the presence of NO/O2. The transmembrane cysteinyl peptides C4 (AcNH-KKACALA(LA)6KK-CONH2) and C8 (AcNH-KKALALACALA(LA)3KK-CONH2) were incorporated into dilauroylphosphatidylcholine bilayers; their location in the membrane was determined by EPR spin labeling. The peptides, C8 and C4, and glutathione (GSH; 300 μM) were treated with a NO donor, DEA-NONOate, and nitrosothiol formation was determined under various O2 levels. Surprisingly, the more hydrophobic cysteinyl peptide, C8, did not yield any S-nitrosated product compared to GSH in the aqueous phase or C4 peptide in the liposomes in the presence of NO/O2. These data suggest that thiols located deeply in the hydrophobic core of the membrane may be less likely to undergo S-nitrosation in the presence of NO/O2.