Ascenzi Paolo, di Masi Alessandra, Coletta Massimo, Ciaccio Chiara, Fanali Gabriella, Nicoletti Francesco P, Smulevich Giulietta, Fasano Mauro
Department of Biology and Interdepartmental Laboratory for Electron Microscopy, University Roma Tre, I-00146 Roma, Italy.
J Biol Chem. 2009 Nov 6;284(45):31006-17. doi: 10.1074/jbc.M109.010736. Epub 2009 Sep 3.
Human serum albumin (HSA) participates in heme scavenging; in turn, heme endows HSA with myoglobin-like reactivity and spectroscopic properties. Here, the allosteric effect of ibuprofen on peroxynitrite isomerization to NO(3)(-) catalyzed by ferric human serum heme-albumin (HSA-heme-Fe(III)) is reported. Data were obtained at 22.0 degrees C. HSA-heme-Fe(III) catalyzes peroxynitrite isomerization in the absence and presence of CO(2); the values of the second order catalytic rate constant (k(on)) are 4.1 x 10(5) and 4.5 x 10(5) m(-1) s(-1), respectively. Moreover, HSA-heme-Fe(III) prevents peroxynitrite-mediated nitration of free added l-tyrosine. The pH dependence of k(on) (pK(a) = 6.9) suggests that peroxynitrous acid reacts preferentially with the heme-Fe(III) atom, in the absence and presence of CO(2). The HSA-heme-Fe(III)-catalyzed isomerization of peroxynitrite has been ascribed to the reactive pentacoordinated heme-Fe(III) atom. In the absence and presence of CO(2), ibuprofen impairs dose-dependently peroxynitrite isomerization by HSA-heme-Fe(III) and facilitates the nitration of free added l-tyrosine; the value of the dissociation equilibrium constant for ibuprofen binding to HSA-heme-Fe(III) (L) ranges between 7.7 x 10(-4) and 9.7 x 10(-4) m. Under conditions where [ibuprofen] is >>L, the kinetics of HSA-heme-Fe(III)-catalyzed isomerization of peroxynitrite is superimposable to that obtained in the absence of HSA-heme-Fe(III) or in the presence of non-catalytic HSA-heme-Fe(III)-cyanide complex and HSA. Ibuprofen binding impairs allosterically peroxynitrite isomerization by HSA-heme-Fe(III), inducing the hexacoordination of the heme-Fe(III) atom. These results represent the first evidence for peroxynitrite isomerization by HSA-heme-Fe(III), highlighting the allosteric modulation of HSA-heme-Fe(III) reactivity by heterotropic interaction(s), and outlining the role of drugs in modulating HSA functions. The present results could be relevant for the drug-dependent protective role of HSA-heme-Fe(III) in vivo.
人血清白蛋白（HSA）参与血红素清除；反过来，血红素赋予HSA类似肌红蛋白的反应活性和光谱特性。在此，报道了布洛芬对由高铁人血清血红素 - 白蛋白（HSA - 血红素 - Fe(III)）催化的过氧亚硝酸根异构化为NO₃⁻的变构效应。数据是在22.0℃下获得的。HSA - 血红素 - Fe(III)在有和没有CO₂存在的情况下均催化过氧亚硝酸根异构化；二级催化速率常数（k(on)）的值分别为4.1×10⁵和4.5×10⁵ m⁻¹ s⁻¹。此外，HSA - 血红素 - Fe(III)可防止过氧亚硝酸根介导的游离添加的L - 酪氨酸的硝化。k(on)的pH依赖性（pK(a) = 6.9）表明，在有和没有CO₂存在的情况下，过氧亚硝酸优先与血红素 - Fe(III)原子反应。HSA - 血红素 - Fe(III)催化的过氧亚硝酸根异构化归因于具有反应活性的五配位血红素 - Fe(III)原子。在有和没有CO₂存在的情况下，布洛芬剂量依赖性地损害HSA - 血红素 - Fe(III)催化的过氧亚硝酸根异构化，并促进游离添加的L - 酪氨酸的硝化；布洛芬与HSA - 血红素 - Fe(III)（L）结合的解离平衡常数的值在7.7×10⁻⁴至9.7×10⁻⁴ m之间。在[布洛芬] >> L的条件下，HSA - 血红素 - Fe(III)催化的过氧亚硝酸根异构化动力学与在没有HSA - 血红素 - Fe(III)或存在非催化性HSA - 血红素 - Fe(III) - 氰化物络合物和HSA的情况下获得的动力学叠加。布洛芬结合变构性地损害HSA - 血红素 - Fe(III)催化的过氧亚硝酸根异构化，诱导血红素 - Fe(III)原子的六配位。这些结果代表了HSA - 血红素 - Fe(III)催化过氧亚硝酸根异构化的首个证据，突出了通过异源相互作用对HSA - 血红素 - Fe(III)反应活性的变构调节，并概述了药物在调节HSA功能中的作用。目前的结果可能与HSA - 血红素 - Fe(III)在体内的药物依赖性保护作用相关。
