Chemiluminescent detection of S-nitrosated proteins: comparison of tri-iodide, copper/CO/cysteine, and modified copper/cysteine methods.

The precise quantification of high and low molecular weight S-nitrosothiols (RSNOs) in biological samples is necessary for the study of nitric oxide-dependent posttranslational signal transduction. Several chemiluminescence-based methods are used for the detection of S-nitrosothiols using the nitric oxide analyzer, including the tri-iodide method and the Cu/CO/cysteine (3C) method. Despite the fact that the tri-idodide method is the most widely used and validated methodology, the levels of S-nitrosated hemoglobin (SNO-Hb) and S-nitrosated albumin have been lower than those reported using photolysis coupled to chemiluminescence. This chapter demonstrates that the tri-iodide method and a newly developed modified copper/cysteine (2C) method compare favorably with the 3C method. Our comparisons include physiologically relevant conditions where the ratio of SNO to heme is low and the frequency of nitrosation of a given Hb tetramer is less than 1. In our studies, the tri-iodide method, the 3C method, and the modified 2C method give consistent and reproducible results. These studies suggest that the proper use of any of these methods can be effective in the accurate measurement of S-nitrosothiols in biological samples. Using more than one in combination has the potential to resolve controversies related to the role of hemoglobin in the generation of RSNOs or the role of RSNOs in biology, whether the RSNOs derive from nitrite or other nitrosative pathways.