Laboratory of Biochemistry and Vascular Biology, Division of Hematology, Center for Biologics Evaluation and Research, US Food and Drug Administration, Bethesda, Maryland 20892, United States.
Biochemistry. 2011 Nov 15;50(45):9752-66. doi: 10.1021/bi200876e. Epub 2011 Oct 21.
We have previously shown that hydrogen peroxide (H(2)O(2)) triggers irreversible oxidation of amino acids exclusive to the β-chains of purified human hemoglobin (HbAo). However, it is not clear, whether α- or β-subunit Hb variants exhibit different oxidative resistance to H(2)O(2) when compared to their native HbAo. Hb Providence contains two β-subunit variants with single amino acid mutations at βLys82→Asp (βK82D) and at βLys82→Asn (βK82N) positions and binds oxygen at lower affinity than wild type HbA. We have separated Hb Providence into its 3 component fractions, and contrasted oxidative reactions of its β-mutant fractions with HbAo. Relative to HbAo, both βK82N and βK82D fractions showed similar autoxidation kinetics and similar initial oxidation reaction rates with H(2)O(2). However, a more profound pattern of changes was seen in HbAo than in the two Providence fractions. The structural changes in HbAo include a collapse of β-subunits, and α-α dimer formation in the presence of excess H(2)O(2). Mass spectrometric and amino acid analysis revealed that βCys93 and βCys112 were oxidized in the HbAo fraction, consistent with oxidative pathways driven by a ferrylHb and its protein radical. These amino acids were oxidized at a lesser extent in βK82D fraction. While the 3 isolated components of Hb Providence exhibited similar ligand binding and oxidation reaction kinetics, the variant fractions were more effective in consuming H(2)O(2) and safely internalizing radicals through the ferric/ferryl pseudoperoxidase cycle.
我们之前已经证明，过氧化氢（H₂O₂）会引发纯化的人血红蛋白（HbAo）β 链上特有的氨基酸不可逆氧化。然而，当与天然 HbAo 相比时，α-或β-亚基 Hb 变体是否表现出对 H₂O₂不同的氧化抗性尚不清楚。Hb Providence 包含两个β亚基变体，在βLys82→Asp（βK82D）和βLys82→Asn（βK82N）位置处单个氨基酸发生突变，与野生型 HbA 相比，其氧结合亲和力较低。我们已经将 Hb Providence 分离成其 3 个组成部分，并对比了其β突变部分与 HbAo 的氧化反应。与 HbAo 相比，βK82N 和βK82D 部分均显示出相似的自氧化动力学和相似的初始 H₂O₂氧化反应速率。然而，在 HbAo 中看到的变化模式比在两个 Providence 部分中更为深刻。HbAo 中的结构变化包括β亚基的崩溃和过量 H₂O₂存在下的α-α二聚体形成。质谱和氨基酸分析表明，βCys93 和βCys112 在 HbAo 部分被氧化，与由铁血红蛋白和其蛋白自由基驱动的氧化途径一致。在βK82D 部分，这些氨基酸被氧化的程度较小。虽然 Hb Providence 的 3 个分离成分表现出相似的配体结合和氧化反应动力学，但变体部分更有效地消耗 H₂O₂并通过三价/四价假过氧化物酶循环安全地内化自由基。
