THE REACTIONS OF 3,5-DIBROMO-4-NITROSOBENZENESULFONATE AND ITS BIOLOGICAL APPLICATIONS

Recently, 5,5-dibromo-4-nitrosobenzenesulfonate (DBNBS) has been applied to detect biological free radicals. However, DBNBS has various non-specific reactions which lead to perplexing results. Thus, we investigated some basic reactions of DBNBS in combination of other nitroso spin traps to assign DBNBS spin adducts derived from human platelets which presumeably related to the endothelium-derived relaxing factor (EDRF). The collagen activated platelets yielded four spin adducts (S(T), L(T), S(S), and L(S)) in the presence of DBNBS (40 mM). The broad triplet due to S(T) was also observed by bubbling NO gas into a DBNBS solution. To identify S(T), nitrosobenzene (NB) in dry dioxane was mixed with NO-saturated dioxane. The NB-NO spin adduct was observed but decomposed into diphenyl aminoxyl by the addition of H2O indicating that the primary adduct formed by the reaction of NO and DBNBS is unstable and turns into a dimerization product. Although S(T) could be eliminated by the inhibitor of EDRF, S(T) was shown to be produced by non-specific reactions. Another triplet was assigned to an S-centered radical because thiyl radicals which were generated from either the decomposition of S-nitrosothiol, or glutathione oxidation exhibited almost identical triplet signals. The other two sextets were assigned to C-centered radical adducts. Thus, DBNBS detected NO-related, S-centered, and two C-centered radicals derived from human platelets. Special cautions are necessary for the identification of DBNBS spin adducts in a biological system to exclude artifactual radicals.