Boys Brian L, Konermann Lars
Department of Chemistry, The University of Western Ontario, London, Ontario, Canada.
J Am Soc Mass Spectrom. 2007 Jan;18(1):8-16. doi: 10.1016/j.jasms.2006.08.013. Epub 2006 Sep 18.
The native structure of hemoglobin (Hb) comprises two alpha- and two beta-subunits, each of which carries a heme group. There appear to be no previous studies that report the in vitro folding and assembly of Hb from highly unfolded alpha- and beta-globin in a "one-pot" reaction. One difficulty that has to be overcome for studies of this kind is the tendency of Hb to aggregate during refolding. This work demonstrates that denaturation of Hb in 40% acetonitrile at pH 10.0 is reversible. A dialysis-mediated solvent change to a purely aqueous environment of pH 8.0 results in Hb refolding without any apparent aggregation. Fluorescence, Soret absorption, circular dichroism, and ESI mass spectra of the protein recorded before unfolding and after refolding are almost identical. By employing an externally pressurized dialysis cell that is coupled on-line to an ESI mass spectrometer, changes in heme binding behavior, protein conformation, and quaternary structure can be monitored as a function of time. The process occurs in a stepwise sequential manner, leading from monomeric alpha- and beta-globin to heterodimeric species, which then assemble into tetramers. Overall, this mechanism is consistent with previous studies employing the mixing of folded alpha- and beta-globin. However, some unexpected features are observed, e.g., a heme-deficient beta-globin dimer that represents an off-pathway intermediate. Monomeric beta-globin is capable of binding heme before forming a complex with an alpha-subunit. This observation suggests that holo-alpha-apo-beta globin does not represent an obligatory intermediate during Hb assembly, as had been proposed previously. The on-line dialysis/ESI-MS approach developed for this work represents a widely applicable tool for studying the folding and self-assembly of noncovalent biological complexes.
血红蛋白（Hb）的天然结构由两个α亚基和两个β亚基组成，每个亚基都携带一个血红素基团。此前似乎没有研究报道过在“一锅法”反应中从高度解折叠的α和β珠蛋白体外折叠和组装Hb。这类研究必须克服的一个困难是Hb在重折叠过程中易于聚集。这项工作表明，Hb在pH 10.0的40%乙腈中变性是可逆的。通过透析介导的溶剂变化至pH 8.0的纯水性环境，可使Hb重折叠而无明显聚集。在解折叠前和解折叠后记录的蛋白质的荧光、Soret吸收、圆二色性和电喷雾电离质谱几乎相同。通过使用与电喷雾电离质谱仪在线连接的外部加压透析池，可以监测血红素结合行为、蛋白质构象和四级结构随时间的变化。该过程以逐步顺序的方式发生，从单体α和β珠蛋白转变为异二聚体，然后组装成四聚体。总体而言，该机制与先前使用折叠的α和β珠蛋白混合的研究一致。然而，观察到一些意外特征，例如，一种血红素缺陷的β珠蛋白二聚体，它代表一条偏离途径的中间体。单体β珠蛋白在与α亚基形成复合物之前能够结合血红素。这一观察结果表明，全α脱辅基β珠蛋白并不像先前提出的那样是Hb组装过程中的一个必需中间体。为本工作开发的在线透析/电喷雾电离质谱方法是研究非共价生物复合物折叠和自组装的一种广泛适用的工具。
