Casey J R, Reithmeier R A
Department of Medicine, University of Toronto, Ontario, Canada.
Biochemistry. 1993 Feb 2;32(4):1172-9. doi: 10.1021/bi00055a023.
The interaction of band 3, the 95-kDa anion-exchange protein of the human erythrocyte membrane, with a variety of nonionic detergents was studied. Band 3 dimers (Stokes radius = 76 A) prepared in octaethylene glycol monododecyl ether (C12E8) could be exchanged into a variety of detergents by size-exclusion high-performance liquid chromatography (HPLC), with complete removal of C12E8 from band 3 being confirmed using radiolabeled detergent. Critical micellar concentration (cmc) values, determined for all detergents in the buffer used for HPLC analysis, ranged from 0.47 microM to 223 mM. Band 3 was found to aggregate in all detergents below their cmc, and concentrations of detergents 2-200 times the cmc were required to prevent aggregation. For detergents with a low cmc, it was important to ensure that the concentration of detergent micelles minimally equalled the concentration of protein. Hydrodynamic measurements and cross-linking studies showed that band 3 remained dimeric in most detergents above their cmc. Furthermore, circular dichroism and inhibitor binding studies supported the view that band 3 can retain its native structure after detergent exchange. Detergents with short alkyl chains (C8) denature band 3, while detergents with longer alkyl chains (C12) maintained the native structure of band 3. The ability to exchange band 3 into a variety of detergents with the maintenance of native structure is an essential prerequisite for crystallization trials. The results obtained in this study of band 3, a model polytopic (multispanning) membrane protein, may be generally applicable to other membrane proteins.
研究了人红细胞膜上95 kDa阴离子交换蛋白带3与多种非离子去污剂的相互作用。在八甘醇单十二烷基醚（C12E8）中制备的带3二聚体（斯托克斯半径 = 76 Å）可通过尺寸排阻高效液相色谱（HPLC）交换到多种去污剂中，使用放射性标记去污剂确认带3中的C12E8已完全去除。在用于HPLC分析的缓冲液中测定的所有去污剂的临界胶束浓度（cmc）值范围为0.47 μM至223 mM。发现带3在所有低于其cmc的去污剂中都会聚集，需要cmc的2 - 200倍浓度的去污剂来防止聚集。对于cmc较低的去污剂，重要的是要确保去污剂胶束的浓度至少等于蛋白质的浓度。流体动力学测量和交联研究表明，在高于其cmc的大多数去污剂中，带3保持二聚体状态。此外，圆二色性和抑制剂结合研究支持这样的观点，即带3在去污剂交换后可以保留其天然结构。具有短烷基链（C8）的去污剂会使带3变性，而具有较长烷基链（C12）的去污剂则保持带3的天然结构。将带3交换到多种去污剂中并保持天然结构的能力是结晶试验的必要前提。在这项对模型多跨膜蛋白带3的研究中获得的结果可能普遍适用于其他膜蛋白。
