Yamaguchi T, Pang J, Reddy K S, Witkowska H E, Surrey S, Adachi K
The Children's Hospital of Philadelphia, Division of Hematology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
J Biol Chem. 1996 Oct 25;271(43):26677-83. doi: 10.1074/jbc.271.43.26677.
Authentic soluble human beta-globin chains were produced in Escherichia coli using an expression plasmid (pHE2beta) containing full-length cDNAs coding for human beta-globin chain and methionine aminopeptidase. Spectral properties of the purified beta-globin were identical to those of authentic beta-globin. Soluble beta-globin showed low (16 kDa) and high molecular mass (32 kDa) forms that could be separated by gel filtration chromatography. SDS-polyacrylamide gel electrophoresis and electrospray mass spectrometry revealed the 32-kDa species was dimeric beta-globin formed by an intermolecular disulfide bond, while the 16-kDa species was authentic monomeric beta-globin. Monomeric forms of beta-globin, like authentic native beta-globin, formed tetrameric hemoglobin (Hb) A (alpha2beta2) in vitro upon incubation with alpha-globin, while dimeric forms did not. When beta-globin dimers, however, were converted to monomers by incubation with dithiothreitol, the beta-globin chain monomers assembled with alpha-globin and formed hemoglobin tetramers. alpha-Globin was more thermally unstable than beta-globin, while assembled tetramers promoted higher stability. Disulfide-bonded beta-globin dimers showed a slight increase in thermal stability compared with beta-globin; however, dimers were still more unstable than tetrameric Hb A. These results indicate that presence of alpha chains favors assembly with beta-globin, beta-beta dimers cannot bind alpha chains, and that Hb A tetramer formation results in the most thermally stable species.
利用含有编码人β-珠蛋白链和甲硫氨酸氨肽酶的全长cDNA的表达质粒（pHE2β），在大肠杆菌中生产出了真实的可溶性人β-珠蛋白链。纯化后的β-珠蛋白的光谱特性与真实的β-珠蛋白相同。可溶性β-珠蛋白呈现出低分子量（16 kDa）和高分子量（32 kDa）两种形式，可通过凝胶过滤色谱法分离。SDS-聚丙烯酰胺凝胶电泳和电喷雾质谱分析表明，32 kDa的物种是由分子间二硫键形成的二聚体β-珠蛋白，而16 kDa的物种是真实的单体β-珠蛋白。β-珠蛋白的单体形式，如同真实的天然β-珠蛋白一样，在与α-珠蛋白一起孵育时，能在体外形成四聚体血红蛋白（Hb）A（α2β2），而二聚体形式则不能。然而，当β-珠蛋白二聚体与二硫苏糖醇一起孵育转化为单体时，β-珠蛋白链单体与α-珠蛋白组装并形成血红蛋白四聚体。α-珠蛋白比β-珠蛋白热稳定性更低，而组装好的四聚体具有更高的稳定性。与β-珠蛋白相比，二硫键连接的β-珠蛋白二聚体的热稳定性略有增加；然而，二聚体仍然比四聚体Hb A更不稳定。这些结果表明，α链的存在有利于与β-珠蛋白组装，β-β二聚体不能结合α链，并且Hb A四聚体的形成产生了热稳定性最高的物种。
