Strader Michael Brad, Bangle Rachel, Parker Siburt Claire J, Varnado Cornelius L, Soman Jayashree, Benitez Cardenas Andres S, Samuel Premila P, Singleton Eileen W, Crumbliss Alvin L, Olson John S, Alayash Abdu I
Laboratory of Biochemistry and Vascular Biology, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993, U.S.A.
Department of Chemistry, Duke University, Durham, NC 27708, U.S.A.
Biochem J. 2017 Dec 11;474(24):4171-4192. doi: 10.1042/BCJ20170491.
Previous work suggested that hemoglobin (Hb) tetramer formation slows autoxidation and hemin loss and that the naturally occurring mutant, Hb Providence (HbProv; βK82D), is much more resistant to degradation by HO We have examined systematically the effects of genetic cross-linking of Hb tetramers with and without the HbProv mutation on autoxidation, hemin loss, and reactions with HO, using native HbA and various wild-type recombinant Hbs as controls. Genetically cross-linked Hb Presbyterian (βN108K) was also examined as an example of a low oxygen affinity tetramer. Our conclusions are: (a) at low concentrations, all the cross-linked tetramers show smaller rates of autoxidation and hemin loss than HbA, which can dissociate into much less stable dimers and (b) the HbProv βK82D mutation confers more resistance to degradation by HO, by markedly inhibiting oxidation of the β93 cysteine side chain, particularly in cross-linked tetramers and even in the presence of the destabilizing Hb Presbyterian mutation. These results show that cross-linking and the βK82D mutation do enhance the resistance of Hb to oxidative degradation, a critical element in the design of a safe and effective oxygen therapeutic.
先前的研究表明，血红蛋白（Hb）四聚体的形成会减缓自氧化和血红素损失，并且天然存在的突变体血红蛋白普罗维登斯（HbProv；βK82D）对HO的降解具有更强的抗性。我们使用天然HbA和各种野生型重组血红蛋白作为对照，系统地研究了有无HbProv突变的Hb四聚体的基因交联对自氧化、血红素损失以及与HO反应的影响。还研究了基因交联的血红蛋白长老会（βN108K）作为低氧亲和力四聚体的一个例子。我们的结论是：（a）在低浓度下，所有交联的四聚体的自氧化和血红素损失速率均低于HbA，HbA可解离为稳定性更低的二聚体；（b）HbProvβK82D突变通过显著抑制β93半胱氨酸侧链的氧化，赋予了对HO降解更强的抗性，特别是在交联的四聚体中，甚至在存在使稳定性降低的血红蛋白长老会突变的情况下也是如此。这些结果表明，交联和βK82D突变确实增强了Hb对氧化降解的抗性，这是安全有效的氧治疗剂设计中的关键要素。
