Edelstein S J, McEwen B, Gibson Q H
J Biol Chem. 1976 Dec 10;251(23):7632-7.
The tetramer-dimer dissociation equilibria (K 4,2) of several fish hemoglobins have been examined by sedimentation velocity measurements with a scanner-computer system for the ultracentrifuge and by flash photolysis measurements using rapid kinetic methods. Samples studied in detail included hemoglobins from a marine teleost, Brevoortia tyrannus (common name, menhaden); a fresh water teleost, Cyprinus carpio, (common name, carp); and an elasmobranch Prionace glauca (common name, blue shark). For all three species in the CO form at pH 7, in 0.1 M phosphate buffer, sedimentation coefficients of 4.3 S (typical of tetrameric hemoglobin) are observed in the micromolar concentration range. In contrast, mammalian hemoglobins dissociate appreciably to dimers under these conditions. The inability to detect dissociation in three fish hemoglobins at the lowest concentrations examined indicates that K 4,2 must have a value of 10(-8) M or less. In flash photolysis experiments on very dilute solutions in long path length cells, two kinetic components were detected with their proportions varying as expected for an equilibrium between tetramers (the slower component) and dimers (the faster component); values of K 4,2 for the three fish hemoglobins in the range 10(-9) to 10(-8) M were calculated from these data. Thus, the values of K 4,2 for liganded forms of the fish hemoglobins appear to be midway between the value for liganded human hemoglobin (K 4,2 approximately 10(-6) M) and unliganded human hemoglobin (K 4,2 approximately 10(-12) M). This conclusion is supported by measurements on solutions containing guanidine hydrochloride to enhance the degree of dissociation. All three fish hemoglobins are appreciably dissociated at guanidine concentrations of about 0.8 M, which is roughly midway between the guanidine concentrations needed to cause comparable dissociation of liganded human hemoglobin (about 0.4 M) and unliganded human hemoglobin (about 1.6 M). Kinetic measurements on solutions containing guanidine hydrochloride indicated that there are changes in both the absolute rates and the proportions of the fast and slow components, which along with other factors complicated the analysis of the data in terms of dissociation constants. Measurements were also made in solutions containing urea to promote dissociation, but with this agent very high concentrations (about 6 M) were required to give measureable dissociation and the fish hemoglobins were unstable under these conditions, with appreciable loss of absorbance spectra in both the sedimentation and kinetic experiments.
通过使用超速离心机的扫描器 - 计算机系统进行沉降速度测量以及采用快速动力学方法进行闪光光解测量，对几种鱼类血红蛋白的四聚体 - 二聚体解离平衡（K₄,₂）进行了研究。详细研究的样本包括一种海洋硬骨鱼——油鲱（学名：Brevoortia tyrannus，俗名：鲱鱼）的血红蛋白；一种淡水硬骨鱼——鲤鱼（学名：Cyprinus carpio，俗名：鲤鱼）的血红蛋白；以及一种软骨鱼——灰鲭鲨（学名：Prionace glauca，俗名：蓝鲨）的血红蛋白。对于所有这三个物种处于pH 7的CO形式、0.1 M磷酸盐缓冲液中的样本，在微摩尔浓度范围内观察到沉降系数为4.3 S（典型的四聚体血红蛋白）。相比之下，哺乳动物血红蛋白在这些条件下会明显解离为二聚体。在所检测的最低浓度下无法检测到三种鱼类血红蛋白的解离，这表明K₄,₂的值必定为10⁻⁸ M或更低。在长光程细胞中对非常稀溶液进行的闪光光解实验中，检测到两个动力学组分，其比例随四聚体（较慢组分）和二聚体（较快组分）之间的平衡预期而变化；根据这些数据计算出三种鱼类血红蛋白的K₄,₂值在10⁻⁹至10⁻⁸ M范围内。因此，鱼类血红蛋白配体形式的K₄,₂值似乎介于配体化人血红蛋白的值（K₄,₂约为10⁻⁶ M）和未配体化人血红蛋白的值（K₄,₂约为10⁻¹² M）之间。通过对含有盐酸胍以增强解离程度的溶液进行测量，这一结论得到了支持。在约0.8 M的盐酸胍浓度下，所有三种鱼类血红蛋白都明显解离，该浓度大致介于使配体化人血红蛋白产生可比解离所需的盐酸胍浓度（约0.4 M）和未配体化人血红蛋白所需的盐酸胍浓度（约1.6 M）之间。对含有盐酸胍的溶液进行的动力学测量表明，快速和慢速组分的绝对速率和比例都发生了变化，这与其他因素一起使根据解离常数对数据进行分析变得复杂。还在含有尿素以促进解离的溶液中进行了测量，但使用这种试剂需要非常高的浓度（约6 M）才能产生可测量的解离，并且在这些条件下鱼类血红蛋白不稳定，在沉降和动力学实验中吸光度光谱都有明显损失。
