Study of the regulation mechanism of ligand affinity of hemoglobin by protein engineering

根据动物物种的生活环境，血红蛋白的氧亲和力被调节在25,000倍的范围内。为了了解蛋白质部分氨基酸序列差异是通过什么机制实现这种调节的，我们利用基于定点突变的蛋白质工程方法合成了8个特定氨基酸取代的人血红蛋白人工突变体，并阐明了它们的氧结合性质等特征，给出了以下结果。His近端突变体：用Leu、Val、Ile、Ala或Phe或His-92β替换为Leu、Val、Ile、Ala或Phe，或用Tyr替换His-92β的6个突变体，表现出氧亲和力变化36倍、IHP(六磷酸肌醇)效应、玻尔效应和协作性降低，以及血红素铁的自氧化增加等氧平衡特性。Thr-38pha突变体：合成了两个突变体，其中参与α1-β2亚基之间氢键形成的Thr-38pha被Ser或Val取代。前者表现出几乎正常的氧结合性质，而后者表现出一些改变的性质(氧亲和力增加2.8倍)。电子光谱、振动光谱和质子核磁共振波谱分析表明，它们的高阶结构正常。目前的实验结果表明，近端残基广泛参与氧亲和力调节，而Thr-38α不参与。他们还表明，近端残基可以实现36倍的氧亲和力调节。然而，与其他研究人员的数据检验表明，仅通过取代血红素基团周围残基的亲和力调节远远不是25,000倍的变化，这表明必须调用其他机制。

The oxygen affinity of hemoglobin is regulated in a range of 25,000-folds depending on the living environment of animal species. To know by what mechanism the regulation is realized from amino acid sequence differences in protein moiety, we synthesized eight artificial mutants of human hemoglobin with particular amino acids replaced by others by means of protein engineering based on site-directed mutagenesis and elucidated their characteristics such as oxygen binding properties, giving the following results.1. Proximal His mutants : Six mutants, in which the heme iron-bound invariable His-87alpha residue is replaced by either Leu, Val, Ile, Ala or Phe, or His-92beta is replaced by Tyr, showed such oxygen equilibrium properties as 36-fold variation of oxygen affinity, various decreases in the effect of IHP(inositol hexaphosphate), the Bohr effect and cooperativity, and increase in autooxidation of heme iron.2. Thr-38alpha mutants : Two mutants in which Thr-38alpha, participating in hydrogen bond formation between the alpha1-beta2 subunits, is replaced by Ser or Val were synthesized. The former showed almost normal oxygen binding properties whereas the latter showed somewhat altered properties (2.8-fold increase in oxygen affinity). The analysis by means of electronic, vibration and proton NMR Spectroscopy indicated that their high order structure is normal.3. The present experimental results indicate that the residues at the proximal side extensively participate in oxygen affinity regualtion whereas Thr-38alpha does not so much. They also indicate that a 36-fold regulation of oxygen affinity can be realized by the proximal residue. However, examination with other investigators' data shows that the affinity reguation by replacements of only the residues surrounding the heme group is far from the 25,000-fold variation, suggesting that some other mechanism must be invoked.