Department of Chemistry, Faculty of Chemistry and Chemical Engineering, Babeş-Bolyai University, 11 Arany Janos Street, 400028 Cluj-Napoca, Romania.
Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark.
Molecules. 2018 Feb 7;23(2):350. doi: 10.3390/molecules23020350.
The autocatalytic reaction between nitrite and the oxy form of globins involves free radicals. For myoglobin (Mb), an initial binding of nitrite to the iron-coordinated oxygen molecule was proposed; the resulting ferrous-peroxynitrate species was not detected, but its decay product, the high-valent ferryl form, was demonstrated in stopped-flow experiments. Reported here are the stopped flow spectra recorded upon mixing oxy Hb (native, as well as chemically-derivatized in the form of several candidates of blood substitutes) with a supraphysiological concentration of nitrite. The data may be fitted to a simple kinetic model involving a transient met-aqua form, in contrast to the ferryl detected in the case of Mb in a similar reaction sequence. These data are in line with a previous observation of a transient accumulation of ferryl Hb under auto-catalytic conditions at much lower concentrations of nitrite (Grubina, R. et al. J. Biol. Chem. 2007, 282, 12916). The simple model for fitting the stopped-flow data leaves a small part of the absorbance changes unaccounted for, unless a fourth species is invoked displaying features similar to the oxy and tentatively assigned as ferrous-peroxynitrate. Density functional theory (DFT) calculations support this latter assignment. The reaction allows for differentiating between the reactivities of various chemically modified hemoglobins, including candidates for blood substitutes. Polymerization of hemoglobin slows the nitrite-induced oxidation, in sharp contrast to oxidative-stress type reactions which are generally accelerated, not inhibited. Sheep hemoglobin is found to be distinctly more resistant to reaction with nitrite compared to bovine Hb, at large nitrite concentrations (stopped-flow experiments directly observing the oxy + nitrite reaction) as well as under auto-catalytic conditions. Copolymerization of Hb with bovine serum albumin (BSA) using glutaraldehyde leads to a distinct increase of the lag time compared to native Hb as well as to any other form of derivatization examined in the present study. The Hb-BSA copolymer also displays a slower initial reaction with nitrite under stopped-flow conditions, compared to native Hb.
亚硝酸盐与球蛋白的氧合形式之间的自动催化反应涉及自由基。对于肌红蛋白（Mb），提出了亚硝酸盐与铁配位氧分子的初始结合；未检测到由此产生的亚铁过硝酸酯物种，但在停流实验中证明了其衰变产物高价铁氧形式。本文报道了混合氧合 Hb（天然，以及以几种血液替代品候选物的化学衍生形式）与超生理浓度亚硝酸盐时记录的停流光谱。数据可以拟合到一个简单的动力学模型，涉及瞬态甲qua 形式，与类似反应序列中 Mb 中检测到的铁氧形式形成对比。这些数据与先前在低得多的亚硝酸盐浓度（Grubina，R.等人，J. Biol. Chem. 2007, 282, 12916）下自动催化条件下铁氧形式瞬态积累的观察结果一致。拟合停流数据的简单模型留下一小部分吸光度变化无法解释，除非引入第四种物种，其特征类似于氧合形式，并暂定为亚铁过硝酸酯。密度泛函理论（DFT）计算支持后一种分配。该反应允许区分各种化学修饰的血红蛋白的反应性，包括血液替代品的候选物。血红蛋白的聚合会减缓亚硝酸盐诱导的氧化，与通常加速而不是抑制的氧化应激型反应形成鲜明对比。与牛 Hb 相比，在大浓度的亚硝酸盐（直接观察 oxy + nitrite 反应的停流实验）以及自动催化条件下，绵羊血红蛋白被发现明显更能抵抗与亚硝酸盐的反应。使用戊二醛将血红蛋白与牛血清白蛋白（BSA）共聚会导致与天然 Hb 以及本研究中检查的任何其他形式的衍生化相比，滞后时间明显增加。与天然 Hb 相比，Hb-BSA 共聚物在停流条件下与亚硝酸盐的初始反应也较慢。
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