Minetti M, Pietraforte D, Carbone V, Salzano A M, Scorza G, Marino G
Laboratorio di Biologia Cellulare, Istituto Superiore di Sanità, Viale Regina Elena 299, I-00161 Roma, Italy.
Biochemistry. 2000 Jun 6;39(22):6689-97. doi: 10.1021/bi9927991.
Peroxynitrite is a strong oxidant involved in cell injury. In tissues, most of peroxynitrite reacts preferentially with CO(2) or hemoproteins, and these reactions affect its fate and toxicity. CO(2) promotes tyrosine nitration but reduces the lifetime of peroxynitrite, preventing, at least in part, membrane crossing. The role of hemoproteins is not easily predictable, because the heme intercepts peroxynitrite, but its oxidation to ferryl species and tyrosyl radical(s) may catalyze tyrosine nitration. The modifications induced by peroxynitrite/CO(2) on oxyhemoglobin were determined by mass spectrometry, and we found that alphaTyr42, betaTyr130, and, to a lesser extent, alphaTyr24 were nitrated. The suggested nitration mechanism is tyrosyl radical formation by long-range electron transfer to ferrylhemoglobin followed by a reaction with ()NO(2). Dityrosine (alpha24-alpha42) and disulfides (beta93-beta93 and alpha104-alpha104) were also detected, but these cross-linkings were largely due to modifications occurring under the denaturing conditions employed for mass spectrometry. Moreover, immunoelectrophoretic techniques showed that the 3-nitrotyrosine content of oxyhemoglobin sharply increased only in molar excess of peroxynitrite, thus suggesting that this hemoprotein is not a catalyst of nitration. The noncatalytic role may be due to the formation of the nitrating species ()NO(2) mainly in molar excess of peroxynitrite. In agreement with this hypothesis, oxyhemoglobin strongly inhibited tyrosine nitration of a target dipeptide (Ala-Tyr) and of membrane proteins from ghosts resealed with oxyhemoglobin. Erythrocytes were poor inhibitors of Ala-Tyr nitration on account of the membrane barrier. However, at the physiologic hematocrit, Ala-Tyr nitration was reduced by 65%. This "sink" function was facilitated by the huge amount of band 3 anion exchanger on the cell membrane. We conclude that in blood oxyhemoglobin is a peroxynitrite scavenger of physiologic relevance.
过氧亚硝酸根是一种参与细胞损伤的强氧化剂。在组织中，大部分过氧亚硝酸根优先与二氧化碳或血红蛋白发生反应，而这些反应会影响其归宿和毒性。二氧化碳促进酪氨酸硝化，但会缩短过氧亚硝酸根的寿命，至少部分地阻止其穿过细胞膜。血红蛋白的作用不易预测，因为血红素会拦截过氧亚硝酸根，但其氧化为高铁血红素和酪氨酸自由基可能会催化酪氨酸硝化。通过质谱法测定了过氧亚硝酸根/二氧化碳对氧合血红蛋白的修饰作用，我们发现α-酪氨酸42、β-酪氨酸130以及程度较轻的α-酪氨酸24发生了硝化。推测的硝化机制是通过向高铁血红蛋白进行远程电子转移形成酪氨酸自由基，随后与（）二氧化氮反应。还检测到了二酪氨酸（α24-α42）和二硫键（β93-β93和α104-α104），但这些交联主要是由于在质谱分析所用的变性条件下发生的修饰。此外，免疫电泳技术表明，只有在过氧亚硝酸根摩尔过量时，氧合血红蛋白的3-硝基酪氨酸含量才会急剧增加，因此表明这种血红蛋白不是硝化的催化剂。这种非催化作用可能是由于主要在过氧亚硝酸根摩尔过量时形成了硝化物种（）二氧化氮。与这一假设一致，氧合血红蛋白强烈抑制目标二肽（丙氨酸-酪氨酸）和用氧合血红蛋白重新封闭的细胞膜蛋白的酪氨酸硝化。由于细胞膜屏障，红细胞对丙氨酸-酪氨酸硝化的抑制作用较弱。然而，在生理血细胞比容下，丙氨酸-酪氨酸硝化减少了65%。细胞膜上大量的带3阴离子交换蛋白促进了这种“汇”功能。我们得出结论，在血液中，氧合血红蛋白是具有生理相关性的过氧亚硝酸根清除剂。
