The βLys66Tyr Variant of Human Hemoglobin as a Component of a Blood Substitute.
The βLys66Tyr Variant of Human Hemoglobin as a Component of a Blood Substitute.
复制标题
人类血红蛋白的 βLys66Tyr 变体作为血液替代品的组成部分。
DOI：
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发表时间：
2016
影响因子：
--
通讯作者：
C. Cooper
中科院分区：
文献类型：
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作者：
R. Silkstone;G. Silkstone;J. A. Bååth;B. Rajagopal;P. Nicholls;B. Reeder;L. Ronda;L. Bulow;C. Cooper
有人提出，将酪氨酸残基引入人血红蛋白（例如βPhe41Tyr）可能能够通过促进内源性和外源性抗氧化剂的远程电子转移，降低细胞外血红蛋白氧载体（HBOC）中铁基血红素物种的毒性。靠近溶剂暴露血红素边缘的表面暴露残留物可能是突变的良好候选者。因此，我们研究了βLys66Tyr突变的性质。加入过氧化氢（H2O2）生成铁基蛋白。与重组野生型（rwt）或βPhe41Tyr相比，抗坏血酸能更快地将βLys66Tyr中的铁基态还原为铁（met）。然而，通过紫外/可见光谱和高效液相色谱分析发现，加入H2O2后，βLys66Tyr遭受了更多的血红素和珠蛋白损伤。βLys66Tyr与rwt蛋白在其他方面存在显著差异。在铁态下，βLys66Tyr形成类似于rwt的氧、一氧化碳和一氧化氮结合的血红素复合物。然而，CO与突变体结合的动力学比rwt更快，表明血红素裂缝更开放。在铁（met）形式中，突变蛋白中没有典型的met Hb酸碱性转变（H2O到-OH）。氰化物结合的双相性也很明显。βLys66Tyr突变体在大肠杆菌中的表达低于rwt蛋白，纯化过程中存在显著的蛋白异质性。虽然βLys66Tyr和rwt以相似的速率自氧化（氧到met），但βLys66Tyr的氧p50非常低。因此，尽管在βLys66Tyr突变体中明显引入了新的电子转移途径，但其异质性和对氧化损伤的易感性使得该突变体不适合作为HBOC的起始材料。
It has been proposed that introducing tyrosine residues into human hemoglobin (e.g. βPhe41Tyr) may be able to reduce the toxicity of the ferryl heme species in extracellular hemoglobin-based oxygen carriers (HBOC) by facilitating long-range electron transfer from endogenous and exogenous antioxidants. Surface-exposed residues lying close to the solvent exposed heme edge may be good candidates for mutations. We therefore studied the properties of the βLys66Tyr mutation. Hydrogen peroxide (H2O2) was added to generate the ferryl protein. The ferryl state in βLys66Tyr was more rapidly reduced to ferric (met) by ascorbate than recombinant wild type (rwt) or βPhe41Tyr. However, βLys66Tyr suffered more heme and globin damage following H2O2 addition as measured by UV/visible spectroscopy and HPLC analysis. βLys66Tyr differed notably from the rwt protein in other ways. In the ferrous state the βLys66Tyr forms oxy, CO, and NO bound heme complexes similar to rwt. However, the kinetics of CO binding to the mutant was faster than rwt, suggesting a more open heme crevice. In the ferric (met) form the typical met Hb acid-alkaline transition (H2O to -OH) appeared absent in the mutant protein. A biphasicity of cyanide binding was also evident. Expression in E. coli of the βLys66Tyr mutant was lower than the rwt protein, and purification included significant protein heterogeneity. Whilst, βLys66Tyr and rwt autoxidised (oxy to met) at similar rates, the oxygen p50 for βLys66Tyr was very low. Therefore, despite the apparent introduction of a new electron transfer pathway in the βLys66Tyr mutant, the heterogeneity, and susceptibility to oxidative damage argue against this mutant as a suitable starting material for a HBOC.
影响因子：
17.3
作者：
Alayash, Abdu I.
通讯作者：
Alayash, Abdu I.