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

根据动物的生存环境，血红蛋白的氧亲和力在2.5万倍的范围内受到调节。为了了解蛋白质片段的氨基酸序列差异是通过何种机制实现调控的，我们利用基于位点定向诱变的蛋白质工程技术合成了8个人血红蛋白特定氨基酸被其他氨基酸取代的人工突变体，并分析了它们的氧结合特性等特点，得到了以下结果：近端His突变体：6个突变体中，与血红素铁结合的不变His- 87α残基被Leu、Val、Ile、Ala或Phe取代，His-92beta被Tyr取代，表现出36倍的氧亲和力变化，IHP（六磷酸肌醇）作用的不同程度降低，玻尔效应和协同性，以及血红素铁的自氧化性增加。Thr-38alpha突变体：合成了两个突变体，其中Thr-38alpha参与alpha1-beta2亚基之间的氢键形成，被Ser或Val取代。前者表现出几乎正常的氧结合特性，而后者表现出一些改变（氧亲和力增加2.8倍）。通过电子、振动和质子核磁共振谱分析表明，它们的高阶结构是正常的。目前的实验结果表明，近端残基广泛参与氧亲和调节，而thr - 38α则不参与。他们还表明，近端残基可以实现36倍的氧亲和调节。然而，与其他研究人员的数据检查表明，仅替换血红素基团周围残基的亲和力调节与25,000倍的变化相差甚远，这表明必须调用一些其他机制。

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.