Nitric oxide (NO) is an important molecule that exerts multiple functions in biological systems. Because of the short-lived nature of NO, S-nitrosothiols (RSNOs) are believed to act as stable NO carriers. Recently, sulfhydryl (SH) containing macromolecules have been shown to be promising NO carriers. In the present study, we aimed to synthesize and characterize a potential NO carrier based on bovine Cu,Zn-superoxide dismutase (bSOD). To prepare S-nitrosated bSOD, the protein was incubated with S-nitrosoglutathione (GSNO) under varied experimental conditions. The results show that significant S-nitrosation of bSOD occurred only at high temperature (50 °C) for prolonged incubation time (>2 h) S-nitrosation efficiency increased with reaction time and reached a plateau at ~4 h. The maximum amount of NO loaded was determined to be about 0.6 mol SNO/mol protein (~30% loading efficiency). The enzymatic activity of bSOD, however, decreased with reaction time. Our data further indicate that NO functionality can only be measured in the presence of extremely high concentrations of Hg2+ or when the protein was denatured by guanidine. Moreover, mildly acidic pH was shown to favor S-nitrosation of bSOD. A model based on unfolding and refolding of bSOD during preparation was proposed to possibly explain our observation.