Abstract

Biologically active nitroxyl (HNO) is one of the least understood nitrogen oxides. It may play a distinct role from NO in protecting the cardiovascular system, and thiols are suspected to be a major nitroxyl target. The major low-molecular-mass intracellular antioxidant, glutathione (GSH), is an important regulator of cellular homeostasis, and is the most likely biological target of HNO. Cysteine (Cys) and HCys (HCys) are naturally occurring thiol-containing amino acids with antioxidant properties and their levels have been linked to many diseases. Reactions of these thiols with Angeli's salt (AS), a HNO donor, were investigated here. N -acetyl-glutathione and N -acetyl-homocysteine were used in this study but are unavailable commercially. An efficient and simple method was developed to prepare N -acetylated low-mass thiols from the corresponding disulfides (e.g., GSSG, homocystine) in aqueous buffer using sulfosuccinimidyl acetate (NHSA) followed by disulfide reduction by immobilized tris (2-carboxyethyl)phosphine (TCEP). The pK a values of the low-mass thiols used here were determined by pH titration in 0.15 M KCl using the GLpKa instrument. GSH was incubated with AS for 30 min and room temperature, and the products were analyzed by ESI-MS. The sulfinamide (GSONH 2 ) and disulfide (GSSG) were formed at pH>5 but GSSG was the dominant product at higher pHs and GSH concentrations. Disulfides only were detected in the incubations of AS with Cys, N -AcCys, HCys, and penicillamine at pH>5. N -acetylation of penicillamine decreased its reactivity with HNO and led to sulfoxide disulfide (RSOSR) formation. Control experiments with NaNO 2 revealed that the products formed in the AS incubates are due to reaction with HNO at pH>5 but with HNO 2 at pH<4, which yields S -nitrosothiols. The results provide the first comparative study of HNO reactivity with biologically important low-mass RSHs. Furthermore, the efficient conversion of GSH to stable GSONH 2 and GSSG products is consistent with the reported depletion of intracellular GSH by HNO. At present, HNO is detected mainly by monitoring its dimerization product, N 2 O. Since N 2 O can arise from other reactions, a specific method of HNO detection is desirable. The high reactivity of thiols with HNO to form sulfinamide was examined as a method of HNO detection. The CysGlu and CysAsp dipeptides, with two negative charges at the C-terminus, exhibited high sulfinamide yields at pH {598}7.4, and are good candidates for the further development of a HNO marker.