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

利用重组DNA进行位点诱变，合成了在特定位点上含有单个氨基酸取代的人造血红蛋白突变体，并对其氧结合功能、光吸收光谱、质子核磁共振（NMR）光谱和共振拉曼散射进行了测定，探讨了被认为是血红蛋白变构特性关键残基的氨基酸残基的作用。合成了4个突变体：Hb Y145betaF，其中Phe取代了被认为在β亚基中引起三级结构变化的tyr145 β；Hb W37betaF，其中Phe取代了与Asp-94alpha在alpha1-beta2界面形成氢键的Trp-37beta；Hb H92betaV和Hb H92betaD，其中近端92 β的His被Val或Asp取代。M13 e阶段。利用大肠杆菌系统将突变体导入人珠蛋白基因，实现了珠蛋白基因的表达。Hb Y145betaF的氧结合功能（氧亲和力、玻尔效应、协同性、六磷酸肌醇效应）变化温和，而Hb W37betaF的变化剧烈。紫外吸收光谱、核磁共振光谱和共振拉曼光谱获得的叔、四元结构数据与功能数据基本一致。我们注意到tyr -145 β的重要作用是其侧链的大小适合于酪氨酸口袋，而不是与asp -94 β形成氢键。Hb W37betaF的剧烈功能变化可能部分归因于部分解离成α二聚体。Hb H92betaV和Hb H92betaD表现出明显的功能改变。中性或带负电荷的残基取代近端His导致变构效应消失，而突变链上的血红素铁维持在铁态，与m型血红蛋白不同。

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.