Institute of Pharmacology, Catholic University School of Medicine, Rome, Italy.
J Cell Mol Med. 2009 Aug;13(8B):2365-75. doi: 10.1111/j.1582-4934.2009.00680.x.
Bilirubin-IX-alpha (BR) is an endogenous molecule with a strong antioxidant feature due to its ability to scavenge free radicals. In this paper, we demonstrated that BR, at concentrations close to those found within the cell (0.1-2.5 microM), acted as a denitrosylating agent and increased the release of nitric oxide from S-nitrosoglutathione (GSNO) and S-nitrosocysteine (SNOC) (2.5 microM). The complexation of BR with saturating concentrations of human serum albumin (HSA, 2.5 microM) did not further increase nitric oxide release from GSNO and SNOC. At concentrations similar to those reached in plasma (5-20 microM), BR denitrosylated S-nitroso-HSA (2.5 microM), the main circulating S-nitrosothiol, and this effect was potentiated by the complexation of BR with saturating HSA (20 microM). Furthermore, the product(s) of the reaction between nitric oxide and BR were identified. Ultraviolet and mass spectrometry analysis revealed that nitric oxide binds to BR forming a N-nitroso derivative (BR-nitric oxide) with extinction coefficients of 1.393 mM(-1)cm(-1) and 2.254 mM(-1)cm(-1) in methanol and NaOH, respectively. The formation of BR-nitric oxide did not occur only in a reconstituted system, but was confirmed in rat fibroblasts exposed to pro-oxidant stimuli. These results provided novel insights on the antioxidant characteristic of BR through its interaction with nitric oxide, a gaseous neurotransmitter with a well-known dual effect, namely neuroprotective under physiological conditions or neurotoxic if produced in excess, and proposed BR-nitric oxide as a new biomarker of oxidative/nitrosative stress.
胆红素-IX-alpha (BR) 是一种内源性分子，具有很强的抗氧化特性，因为它能够清除自由基。在本文中，我们证明 BR 在接近细胞内浓度（0.1-2.5 microM）时，作为一种脱亚硝基剂，增加了从 S-亚硝基谷胱甘肽 (GSNO) 和 S-亚硝基半胱氨酸 (SNOC) (2.5 microM) 的一氧化氮释放。BR 与饱和浓度的人血清白蛋白 (HSA，2.5 microM) 的络合并没有进一步增加 GSNO 和 SNOC 从一氧化氮的释放。在类似于血浆中达到的浓度（5-20 microM）时，BR 使 S-亚硝基-HSA（2.5 microM）脱亚硝基化，这是主要的循环 S-亚硝基硫醇，并且 BR 与饱和 HSA（20 microM）的络合增强了这种作用。此外，还确定了一氧化氮与 BR 反应的产物。紫外和质谱分析表明，一氧化氮与 BR 结合形成 N-亚硝基衍生物（BR-一氧化氮），在甲醇和 NaOH 中的消光系数分别为 1.393 mM(-1)cm(-1)和 2.254 mM(-1)cm(-1)。BR-一氧化氮的形成不仅发生在重组系统中，而且在暴露于促氧化剂刺激的大鼠成纤维细胞中得到了证实。这些结果通过 BR 与一氧化氮的相互作用提供了对 BR 抗氧化特性的新见解，一氧化氮是一种具有众所周知的双重作用的气态神经递质，即在生理条件下具有神经保护作用，如果过量产生则具有神经毒性，并提出 BR-一氧化氮作为氧化/硝化应激的新生物标志物。
