ABSTRACT

The β subunit substitutions, F41Y and K82D, in sickle cell hemoglobin (Hb) (βE6 V) provides significant resistance to oxidative stress by shielding βCys93 from the oxidizing ferryl heme. We evaluated the oxidative resistance of βCys93 to hydrogen peroxide (H₂O₂) in α subunit mutations in βE6 V (at both the putative and lateral contact regions) that included (1) αH20Q/βE6 V; (2) αH50Q/βE6 V; (3) αH20Q/H50Q/βE6 V; (4) αH20R/βE6 V; and (5) αH20R/H50Q/βE6 V. Estimation by mass spectrometry of irreversible oxidation of βCys93 to cysteic acid (CA) was unchanged or moderately increased in the single mutants harboring a H20Q or H50Q substitution when compared to control (βE6 V). The introduction of Arg (R) singularly or in combination with Q enhanced the pseudoperoxidative cycle by slightly decreasing the ferryl in favor of ferrous and ferric species after treatment with H₂O₂. Higher rates for heme loss from the ferric forms of the Q species to the receptor high affinity recombinant apomyglobin were observed in contrast to the R mutants and control. Because of their improved solubility, a combination of Q and R substitutions together with mutations carrying redox active variants (F41Y/K82D) may provide dual antioxidant and antisickling targets in the design of gene therapy-based candidates.

摘要

镰状细胞血红蛋白 (Hb) (βE6 V) 中的 β 亚基取代 F41Y 和 K82D 通过保护 βCys93 免受氧化性铁血红素的影响，提供了对氧化应激的显着抵抗。我们评估了 βCys93 对过氧化氢 (H ₂ O ₂) βE6 V 中的 α 亚基突变（在推定和横向接触区域），包括 (1) αH20Q/βE6 V；(2) αH50Q/βE6 V；(3) αH20Q/H50Q/βE6 V；(4) αH20R/βE6 V；(5) αH20R/H50Q/βE6 V。与对照 (βE6 V) 相比，在携带 H20Q 或 H50Q 取代的单个突变体中，通过质谱法估计 βCys93 不可逆氧化成半胱氨酸 (CA) 没有变化或适度增加。在用 H ₂ O ₂处理后，Arg (R) 的单独引入或与 Q 组合的引入通过略微降低铁离子有利于亚铁和三价铁物种来增强假过氧化循环. 与 R 突变体和对照相比，观察到从 Q 物种的三价铁形式到受体高亲和力重组肌红蛋白的血红素损失率更高。由于它们的溶解度提高，Q 和 R 取代与携带氧化还原活性变体 (F41Y/K82D) 的突变的组合可以在基于基因治疗的候选物设计中提供双重抗氧化和抗镰刀化靶标。