Ikeda-Saito M, Yamamoto H, Imai K, Kayne F J, Yonetani T
J Biol Chem. 1977 Jan 25;252(2):620-4.
Human hemoglobin containing cobalt protoporphyrin IX or cobalt hemoglobin has been separated into two functionally active alpha and beta subunits using a new method of subunit separation, in which the -SH groups of the isolated subunits were successfully regenerated by treatment with dithiothreitol in the presence of catalase. Oxygen equilibria of the isolated subunit chains were examined over a wide range of temperature using Imai's polarographic method (Imai, K., Morimoto, H., Kotani, M., Watari, H., and Kuroda, M. (1970) Biochim. Biophys. Acta 200, 189-196). Kinetic properties of their reversible oxygenation were investigated by the temperature jump relaxation method at 16 degrees. Electron paramagnetic resonance characteristics of the molecules in both deoxy and oxy states were studies at 77K. The oxygen affinity of the individual regenerated chains was higher than that of the tetrameric cobalt hemoglobin and was independent of pH. The enthalpy changes of the oxygenation have been determined as -13.8 kcal/mol and -16.8 kcal/mol for the alpha and beta chains, respectively. The rates of oxygenation were similar to those reported for iron hemoglobin chains, whereas those of deoxygenation were about 10(2) times larger. The effects of metal substitution on oxygenation properties of the isolated chains were correlated with the results obtained previously on cobalt hemoglobin and cobalt myoglobin. The EPR spectrum of the oxy alpha chain showed a distinctly narrowed hyperfine structure in comparison with that of the oxy beta chain, indicating that the environment around the paramagnetic center (the bound oxygen) is different between these chains. In the deoxy form, EPR spectra of alpha and beta chains were indistinguishable. These observations suggest that one of the inequivalences between alpha and beta chains might exist near the distal histidine group.
含钴原卟啉IX的人血红蛋白或钴血红蛋白已通过一种新的亚基分离方法被分离为两个具有功能活性的α和β亚基，在该方法中，分离出的亚基的-SH基团在过氧化氢酶存在下用二硫苏糖醇处理后成功再生。使用今井的极谱法（今井，K.，森本，H.，小谷，M.，渡里，H.，和黑田，M.（1970年）生物化学与生物物理学报200，189 - 196）在很宽的温度范围内研究了分离出的亚基链的氧平衡。在16℃下通过温度跳跃弛豫法研究了它们可逆氧合的动力学性质。在77K下研究了脱氧和氧合状态下分子的电子顺磁共振特性。各个再生链的氧亲和力高于四聚体钴血红蛋白的氧亲和力，并且与pH无关。α链和β链氧合的焓变分别测定为-13.8千卡/摩尔和-16.8千卡/摩尔。氧合速率与报道的铁血红蛋白链的氧合速率相似，而脱氧速率大约大10²倍。金属取代对分离链的氧合性质的影响与先前在钴血红蛋白和钴肌红蛋白上获得的结果相关。与氧合β链相比，氧合α链的电子顺磁共振谱显示出明显变窄的超精细结构，表明这些链中顺磁中心（结合的氧）周围的环境不同。在脱氧形式下，α链和β链的电子顺磁共振谱无法区分。这些观察结果表明α链和β链之间的不等价性之一可能存在于远端组氨酸基团附近。
