Yanase H, Cahill S, Martin de Llano J J, Manning L R, Schneider K, Chait B T, Vandegriff K D, Winslow R M, Manning J M
Rockefeller University, New York, New York 10021.
Protein Sci. 1994 Aug;3(8):1213-23. doi: 10.1002/pro.5560030807.
Site-directed mutagenesis of an important subunit contact site, Asp-99(beta), by a Lys residue (D99K(beta)) was proven by sequencing the entire beta-globin gene and the mutant tryptic peptide. Oxygen equilibrium curves of the mutant hemoglobin (Hb) (2-15 mM in heme) indicated that it had an increased oxygen affinity and a lowered but significant amount of cooperativity compared to native HbA. However, in contrast to normal HbA, oxygen binding of the recombinant mutant Hb was only marginally affected by the allosteric regulators 2,3-diphosphoglycerate or inositol hexaphosphate and was not at all responsive to chloride. The efficiency of oxygen binding by HbA in the presence of allosteric regulators was limited by the mutant Hb. At concentrations of 0.2 mM or lower in heme, the mutant D99K(beta) Hb was predominantly a dimer as demonstrated by gel filtration, haptoglobin binding, fluorescence quenching, and light scattering. The purified dimeric recombinant Hb mutant exists in 2 forms that are separable on isoelectric focusing by about 0.1 pH unit, in contrast to tetrameric hemoglobin, which shows 1 band. These mutant forms, which were present in a ratio of 60:40, had the same masses for their heme and globin moieties as determined by mass spectrometry. The elution positions of the alpha- and beta-globin subunits on HPLC were identical. Circular dichroism studies showed that one form of the mutant Hb had a negative ellipticity at 410 nm and the other had positive ellipticity at this wavelength. The findings suggest that the 2 D99K(beta) recombinant mutant forms have differences in their heme-protein environments.
通过对整个β-珠蛋白基因和突变体胰蛋白酶肽段进行测序，证实了用赖氨酸残基对重要的亚基接触位点天冬氨酸-99（β）进行定点诱变（D99K（β））。突变型血红蛋白（Hb）（血红素浓度为2 - 15 mM）的氧平衡曲线表明，与天然HbA相比，其氧亲和力增加，协同性降低但仍有显著协同性。然而，与正常HbA不同，重组突变型Hb的氧结合仅受到别构调节剂2,3-二磷酸甘油酸或肌醇六磷酸的轻微影响，对氯离子完全无反应。在别构调节剂存在的情况下，HbA的氧结合效率受到突变型Hb的限制。在血红素浓度为0.2 mM或更低时，通过凝胶过滤、触珠蛋白结合、荧光猝灭和光散射证明，突变型D99K（β）Hb主要为二聚体。纯化的二聚体重组Hb突变体以两种形式存在，在等电聚焦上可分离约0.1个pH单位，而四聚体血红蛋白则显示一条带。这些以60:40比例存在的突变形式，通过质谱测定，其血红素和珠蛋白部分的质量相同。α-和β-珠蛋白亚基在HPLC上的洗脱位置相同。圆二色性研究表明，一种形式的突变型Hb在410 nm处具有负椭圆率，另一种在该波长处具有正椭圆率。这些发现表明，两种D99K（β）重组突变形式在其血红素-蛋白质环境中存在差异。
