Yamashiro D J, Adachi M, Konitzer P, Surrey S, Adachi K
Children's Hospital of Philadelphia, Division of Hematology, University of Pennsylvania School of Medicine 19104.
J Biol Chem. 1994 Sep 30;269(39):23996-9.
A recombinant hemoglobin containing Val beta 7 (Hb beta E7V) was engineered and expressed in yeast to evaluate amino acid specificity of the Glu beta 6-->Val mutation (Hb beta E6V) in promoting polymer formation of deoxyhemoglobin. The purified CO Hb beta E7V migrated as a single band on electrophoresis with a slightly decreased positive charge compared with CO Hb S. The oxygen affinity of Hb beta E7V was slightly higher than Hb S, while the absorption spectrum of the mutant was similar to Hb S. Critical concentrations for polymerization in 1.8 M phosphate of the deoxy forms of Hb beta E7V and Hb A were 15- and 25-fold, respectively, higher than Hb S. Oversaturated deoxy Hb beta E7V polymerized without a delay time prior to polymerization like deoxy Hb beta E6F and Hb beta E6W. These results demonstrate that Val beta 6 in Hb S is critical for rapid polymerization of deoxyhemoglobin. The oxy form of Hb beta E7V was approximately 2-3-fold more unstable to heat and mechanical agitation than oxy Hb S, suggesting that instability and polymerization of hemoglobin are distinct properties.
构建了一种含有β链7位缬氨酸（HbβE7V）的重组血红蛋白，并在酵母中进行表达，以评估β链6位谷氨酸突变为缬氨酸（HbβE6V）在促进脱氧血红蛋白聚合物形成方面的氨基酸特异性。纯化后的CO HbβE7V在电泳时呈现为单一条带，与CO Hb S相比，其正电荷略有减少。HbβE7V的氧亲和力略高于Hb S，而该突变体的吸收光谱与Hb S相似。在1.8 M磷酸盐中，HbβE7V和Hb A的脱氧形式发生聚合的临界浓度分别比Hb S高15倍和25倍。过饱和的脱氧HbβE7V像脱氧HbβE6F和HbβE6W一样，在聚合前没有延迟时间就发生了聚合。这些结果表明，Hb S中的β链6位缬氨酸对于脱氧血红蛋白的快速聚合至关重要。HbβE7V的氧合形式对热和机械搅拌的稳定性比氧合Hb S大约低2 - 3倍，这表明血红蛋白的不稳定性和聚合是不同的特性。
