The ability of hemoglobin (Hb) to transport respiratory gases is directly linked to its quaternary structure properties and reversible changes between T (tense) and R (relax) state. In this study we demonstrated that packed red blood cells (pRBCs) storage resulted in a gradual increase in the irreversible changes in the secondary and quaternary structures of Hb, with subsequent impairment of the T↔R transition. Such alteration was associated with the presence of irreversibly settled in the relaxed form, quaternary structure of Hb, which we termed R′. On the secondary structure level, disordered protein organization involved formation of β-sheets and a decrease in α-helices related to the aggregation process stabilized by strong intermolecular hydrogen bonding. Compensatory changes in RBCs metabolism launched to preserve reductive microenvironment were disclosed as an activation of nicotinamide adenine dinucleotide phosphate (NADPH) production and increased reduced to oxidized glutathione (GSH/GSSG) ratio. For the first time we showed the relationship between secondary structure changes and the occurrence of newly discovered R′, which through an artificial increase in oxyhemoglobin level altered Hb ability to bind and release oxygen.

血红蛋白（Hb）输送呼吸气体的能力与其四级结构性质以及T（紧张）状态和R（松弛）状态之间的可逆变化直接相关。在这项研究中，我们证明了堆积的红细胞（pRBC）储存导致Hb二级和四级结构不可逆变化的逐渐增加，并随后损害T↔R转变。这种改变与Hb的四元结构的松弛形式不可逆地沉淀有关，我们称之为R'。在二级结构水平上，无序的蛋白质组织涉及β-折叠的形成和与由强分子间氢键稳定的聚集过程相关的α-螺旋的减少。据披露，为保持还原性微环境而启动的RBC代谢的补偿性变化是通过激活烟酰胺腺嘌呤二核苷酸磷酸（NADPH）产生的，并降低了氧化型谷胱甘肽（GSH / GSSG）的比例。我们首次展示了二级结构变化与新发现的R'之间的关系，后者通过人为增加的氧合血红蛋白水平改变了Hb结合和释放氧的能力。