Is ascorbate able to reduce disulfide bridges? A cautionary note.

The biotin switch assay has recently been proposed as the eligible method to identify different S-nitrosated proteins in biological matrices. However, notwithstanding its wide application, a thorough validation of this method is still lacking. In particular, it has been suggested that ascorbate concentrations higher than 1mM (as proposed in the original method) are needed since ascorbate reaction with S-nitrosothiols is slow. But the selectivity of ascorbate as a cleaving agent of the S-N bond under these conditions has not been well characterized. Our data indicate that ascorbate is able to reduce disulfide bridges of DTNB, cystine, cystinylglycine, glutathione disulfide, protein mixed disulfides and biotin-HPDP with pH and concentration dependent rates. Additionally, we tested the effect of indirect sunlight on ascorbate-mediated cleavage of both disulfides and S-nitrosothiols.