Martin de Llano J J, Manning J M
Rockefeller University, New York, New York 10021.
Protein Sci. 1994 Aug;3(8):1206-12. doi: 10.1002/pro.5560030806.
A recombinant double mutant of hemoglobin (Hb), E6V/L88A(beta), was constructed to study the strength of the primary hydrophobic interaction in the gelation of sickle Hb, i.e., that between the mutant Val-6(beta) of one tetramer and the hydrophobic region between Phe-85(beta) and Leu-88(beta) on an adjacent tetramer. Thus, a construct encoding the donor Val-6(beta) of the expressed recombinant HbS and a second mutation encoding an Ala in place of Leu-88(beta) was assembled. The doubly mutated beta-globin gene was expressed in yeast together with the normal human alpha-chain, which is on the same plasmid, to produce a soluble Hb tetramer. Characterizations of the Hb double mutant by mass spectrometry, by HPLC, and by peptide mapping of tryptic digests of the mutant beta-chain were consistent with the desired mutations. The absorption spectra in the visible and the ultraviolet regions were practically superimposable for the recombinant Hb and the natural Hb purified from human red cells. Circular dichroism studies on the overall structure of the recombinant Hb double mutant and the recombinant single mutant, HbS, showed that both were correctly folded. Functional studies on the recombinant double mutant indicated that it was fully cooperative. However, its gelation concentration was significantly higher than that of either recombinant or natural sickle Hb, indicating that the strength of the interaction in this important donor-acceptor region in sickle Hb was considerably reduced even with such a conservative hydrophobic mutation.
构建了一种血红蛋白（Hb）的重组双突变体E6V/L88A（β），以研究镰状Hb凝胶化过程中主要疏水相互作用的强度，即一个四聚体的突变体Val-6（β）与相邻四聚体上Phe-85（β）和Leu-88（β）之间的疏水区域之间的相互作用强度。因此，组装了一个编码所表达的重组HbS供体Val-6（β）的构建体以及一个将Leu-88（β）替换为Ala的第二个突变体。将双突变的β-珠蛋白基因与位于同一质粒上的正常人α链一起在酵母中表达，以产生可溶性Hb四聚体。通过质谱、高效液相色谱以及对突变β链胰蛋白酶消化产物的肽图谱分析对Hb双突变体进行的表征与预期突变一致。重组Hb与从人红细胞中纯化的天然Hb在可见光和紫外光区域的吸收光谱几乎完全重叠。对重组Hb双突变体和重组单突变体HbS的整体结构进行的圆二色性研究表明，两者均正确折叠。对重组双突变体的功能研究表明它具有完全的协同性。然而，其凝胶化浓度显著高于重组或天然镰状Hb，这表明即使进行了这种保守的疏水突变，镰状Hb中这个重要的供体-受体区域的相互作用强度也大大降低了。
