Kelman D J, Mason R P
Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Arch Biochem Biophys. 1993 Nov 1;306(2):439-42. doi: 10.1006/abbi.1993.1535.
The characterization of the radical formed from rat hemoglobin (Hb) by methemoglobin-generating agents and trapped by 5,5-dimethyl-1-pyrroline N-oxide (DMPO) has shown it to be a thiyl radical. The two-electron oxidation of hemoglobin forms a ferryl species with one or more free radicals located on the globin moiety. While the radical species has not been observed by use of uv-visible spectrophotometry, the species can be detected by use of electron paramagnetic resonance spectroscopy (EPR). In previous studies, in vitro experiments have shown that the EPR signal from the rat Hb radical adduct formed by t-butyl hydroperoxide decreased following pretreatment of the oxyHb with thiol-blocking agents except for iodoacetamide. In this study the power saturation profile of the DMPO radical adduct obtained from the reaction of rat oxyHb with phenylhydrazine exhibited a pattern similar to that obtained from human oxyHb, in which the beta-93 cysteine was labeled with 2,2,6,6-tetramethyl-1-piperidinyloxy-4-maleimide (4-maleimide-TEMPO). EPR spectra were taken at 77 K and computer simulations were performed. The calculated value for a(iso)N obtained by simulation indicates that the radical adduct is in a hydrophobic region. The value for a(iso)H has little structural significance, as the steric effect of the protein makes comparison with radical adducts in solution problematical. The value of gx from the rat Hb radical adduct was significantly higher than that obtained from bovine Hb, whose radical is not thiol-derived, as demonstrated by negative thiol-blocking agent experiments. A higher gx value is consistent with the radical adduct containing a heavy atom such as sulfur. Rat Hb was analyzed for thiol content by use of iodoacetamide, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and uv-visible spectrophotometry. It was found that 1.15 +/- 0.34 thiols/tetramer of Hb were reactive with DTNB, but not iodoacetamide.
由高铁血红蛋白生成剂形成并被5,5-二甲基-1-吡咯啉N-氧化物（DMPO）捕获的大鼠血红蛋白（Hb）自由基的特征表明它是一个硫自由基。血红蛋白的双电子氧化形成一个高铁血红素物种，在球蛋白部分有一个或多个自由基。虽然通过紫外可见分光光度法未观察到自由基物种，但可以通过电子顺磁共振光谱（EPR）检测到该物种。在先前的研究中，体外实验表明，除碘乙酰胺外，用硫醇阻断剂预处理氧合血红蛋白后，由叔丁基过氧化氢形成的大鼠Hb自由基加合物的EPR信号降低。在本研究中，大鼠氧合血红蛋白与苯肼反应得到的DMPO自由基加合物的功率饱和曲线呈现出与人类氧合血红蛋白相似的模式，其中β-93半胱氨酸用2,2,6,6-四甲基-1-哌啶氧基-4-马来酰亚胺（4-马来酰亚胺-TEMPO）标记。在77K下采集EPR光谱并进行计算机模拟。通过模拟得到的a(iso)N计算值表明自由基加合物处于疏水区域。a(iso)H的值几乎没有结构意义，因为蛋白质的空间效应使得与溶液中的自由基加合物进行比较存在问题。大鼠Hb自由基加合物的gx值显著高于牛Hb的gx值，硫醇阻断剂阴性实验表明牛Hb的自由基不是硫醇衍生的。较高的gx值与含有硫等重原子的自由基加合物一致。用碘乙酰胺、5,5'-二硫代双（2-硝基苯甲酸）（DTNB）和紫外可见分光光度法分析大鼠Hb的硫醇含量。发现每四聚体Hb中有1.15±0.34个硫醇与DTNB反应，但不与碘乙酰胺反应。
