Ichimori K, Arroyo C M, Prónai L, Fukahori M, Nakazawa H
Department of Physiology 2, Tokai University, School of Medicine, Kanagawa, Japan.
Free Radic Res Commun. 1993;19 Suppl 1:S129-39. doi: 10.3109/10715769309056s129.
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 preplexing 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 presumably related to the endothelium-derived relaxing factor (EDRF). The collagen activated platelets yielded four spin adducts (ST, LT, SS, and LS) in the presence of DBNBS (40 mM). The broad triplet due to ST was also observed by bubbling NO gas into a DBNBS solution. To identify ST, 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 ST could be eliminated by the inhibitor of EDRF, ST 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 NQ-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.
最近，5,5-二溴-4-亚硝基苯磺酸盐（DBNBS）已被应用于检测生物自由基。然而，DBNBS存在各种非特异性反应，导致结果复杂。因此，我们结合其他亚硝基自旋捕获剂研究了DBNBS的一些基本反应，以确定源自人血小板的DBNBS自旋加合物，这些加合物可能与内皮衍生舒张因子（EDRF）有关。在DBNBS（40 mM）存在下，胶原激活的血小板产生了四种自旋加合物（ST、LT、SS和LS）。通过将NO气体鼓泡到DBNBS溶液中也观察到了由于ST产生的宽三重峰。为了鉴定ST，将干燥二氧六环中的亚硝基苯（NB）与NO饱和的二氧六环混合。观察到了NB-NO自旋加合物，但加入H2O后它分解为二苯基氨氧基，这表明由NO和DBNBS反应形成的初级加合物不稳定，会变成二聚产物。尽管ST可以被EDRF抑制剂消除，但ST被证明是由非特异性反应产生的。另一个三重峰被归为以S为中心的自由基，因为由S-亚硝基硫醇分解或谷胱甘肽氧化产生的硫自由基表现出几乎相同的三重峰信号。另外两个六重峰被归为以C为中心的自由基加合物。因此，DBNBS检测到了源自人血小板的与NQ相关、以S为中心和两个以C为中心的自由基。在生物系统中鉴定DBNBS自旋加合物时需要特别小心，以排除人为产生的自由基。
