Adachi K, Konitzer P, Kim J, Welch N, Surrey S
Children's Hospital of Philadelphia, Department of Pediatrics, Pennsylvania 19104.
J Biol Chem. 1993 Oct 15;268(29):21650-6.
Valine, leucine, tryptophan, and phenylalanine substitutions at the beta 6 position of hemoglobin (Hb) were made using a yeast expression system coupled with a polymerase chain reaction-based mutagenesis strategy. The oxygen affinity and absorption spectra of these mutants were similar to recombinant Hb A except for Hb beta E6W which had a higher absorbance at approximately 280 nm. The deoxy forms of Hb beta E6L and Hb S showed characteristic delay times prior to polymerization. Tetrameric deoxy-Hbs containing tryptophan or phenylalanine at the beta 6 position had higher solubilities and polymerized less readily compared with deoxy-Hb S. However, when oversaturated, these Hbs polymerized without a delay time. These results suggest that Hb beta E6W and Hb beta E6F form polymers upon deoxygenation by a linear polymerization mechanism without nuclei formation. During polymerization, bulky hydrophobic amino acids, like phenylalanine and tryptophan at the beta 6 position, might interact with the acceptor pocket on the surface of an adjacent Hb molecule but may not be able to form stable hydrophobic interactions like beta 6 valine and leucine. Difficulty in insertion of the bulky side chains of these aromatic amino acids into the hydrophobic acceptor pocket on an adjacent tetramer may inhibit nuclei formation prior to polymerization.
利用酵母表达系统结合基于聚合酶链反应的诱变策略，在血红蛋白（Hb）的β6位点进行缬氨酸、亮氨酸、色氨酸和苯丙氨酸取代。除了HbβE6W在约280nm处具有较高吸光度外，这些突变体的氧亲和力和吸收光谱与重组Hb A相似。HbβE6L和Hb S的脱氧形式在聚合前显示出特征性延迟时间。与脱氧Hb S相比，在β6位点含有色氨酸或苯丙氨酸的四聚体脱氧Hb具有更高的溶解度且聚合较不易发生。然而，当过度饱和时，这些Hb无延迟时间地聚合。这些结果表明，HbβE6W和HbβE6F在脱氧时通过线性聚合机制形成聚合物，而不形成核。在聚合过程中，β6位点的苯丙氨酸和色氨酸等庞大疏水氨基酸可能与相邻Hb分子表面的受体口袋相互作用，但可能无法形成像β6缬氨酸和亮氨酸那样稳定的疏水相互作用。这些芳香族氨基酸庞大侧链难以插入相邻四聚体上的疏水受体口袋，可能会抑制聚合前的核形成。
