Studies on the Mechanism of Physiological Function of Hemoglobin by Using Artificial Mutants

利用重组DNA通过定点诱变合成了在特定位点含有单个氨基酸取代的人工血红蛋白突变体，并对其氧结合功能、光吸收光谱、质子核磁共振通过红外光谱和共振拉曼散射研究了血红蛋白变构的关键氨基酸残基的作用。合成：Hb Y145 β F，其中Phe取代Tyr-145 β，被认为诱导β亚基的三级结构变化; Hb W37 β F，其中Phe取代Trp-37 β，其在α 1-β 2界面与Asp-94 α形成氢键; Hb H92 β V和Hb H92 β D，其中92 β处的近端His被瓦尔或Asp取代。M13 Phase-E将突变体导入人珠蛋白基因并在大肠杆菌中表达，结果表明，血红蛋白Y145 β F的氧结合功能（氧亲和力、玻尔效应、协同效应、肌醇六磷酸效应）发生了中等程度的变化，而血红蛋白W37 β F则发生了剧烈的变化。从紫外光吸收、核磁共振和共振拉曼光谱获得的三级和四级结构数据与功能数据几乎一致。值得注意的是，Tyr-145 β的重要作用是其具有大小适合于酪氨酸口袋的侧链，而不是其与Asp-94 β形成氢键。Hb W37 β F的功能变化可能部分归因于部分解离成α二聚体，Hb H92 β V和Hb H92 β D的功能变化也很大。近端His被中性或带负电荷的残基取代导致变构效应消失，而突变链的血红素铁保持在亚铁态，不同于M型血红蛋白。

Artificial hemoglobin mutants which contained single amino acid substitutions at particular sites were synthesized by site-directed mutagenesis using recombinant DNA and their oxygen binding function, light absorption spectra, proton nuclear magnetic resonance (NMR) spectra and resonance Raman scattering were measured to explore the roles of amino acid residues which are considered to be a key residue for the allosteric properties of hemoglobin.Four mutants were synthesized : Hb Y145betaF in which Phe substitutes for Tyr-145beta which is considered to induce a tertiary structure change in the beta subunit ; Hb W37betaF in which Phe substitutes for Trp-37beta which forms a hydrogen bond with Asp-94alpha at the alpha1-beta2 interface ; Hb H92betaV and Hb H92betaD in which the proximal His at 92beta is replaced by Val or Asp. The M13 Phase-E. coli system was used to introduce the mutations into human globin gene and to express the globin gene.The changes in oxygen binding functions (oxygen affinity, the Bohr effect, co-operativity, effect of inositol hexaphosphate) of Hb Y145betaF were moderate while those of Hb W37betaF were drastic. The tertiary and quaternary structure data acquired from UV light absorption, NMR, and resonance Raman spectra were nearly consistent with the functional data. It was noted that the important role of Tyr-145beta is that it has a side chain whose size is appropriate to fit to the tyrosine pocket rather than that it forms a hydrogen bond with Asp-94beta. The drastic functional changes in Hb W37betaF may partly be attributed to partial dissociation into alphabeta dimers.Hb H92betaV and Hb H92betaD showed drastic functional changes. The replacement of the proximal His by neutral or negatively charged residue caused disapearance of allosteric effects whereas the heme iron of the mutant chains were maintained in a ferrous state, different from the M-type hemoglobins.