Two-dimensional 1H NMR studies of cytochrome c: assignment of the N-terminal helix.
Two-dimensional 1H NMR studies of cytochrome c: assignment of the N-terminal helix.
复制标题
细胞色素 c 的二维 1H NMR 研究：N 末端螺旋的分配。
DOI：
10.1021/bi00353a024
复制
发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Englander,SW
中科院分区：
文献类型：
--
作者：
Wand,AJ;Englander,SW
材料与方法马心细胞色素c从Sigma化学公司获得，纯度最高。冻干后的蛋白根据需要溶解在99.8% D20或90% H2O/10% D20的50mm磷酸钾缓冲液中。分配工作中使用的样品用3-5等量的固体二亚硫酸钠还原，根据需要用稀释的NaOD或DC1调节到pH* 5.7，并在长时间的数据采集期间保持在氮下。D20溶液中蛋白质浓度为6 ~ 8mm， H20溶液中蛋白质浓度为12 ~ 14mm。D20样品在pH* 6和室温下与氧化态溶剂交换10分钟后进行还原。二维核磁共振波谱记录在布鲁克wm500光谱仪（耶鲁大学，纽黑文和华盛顿大学，西雅图）上。/-相关（COSY）光谱用标准脉冲序列记录（Aue et al., 1976; Nagayama et al., 1979; Bax & Freeman, 1981）
Materials and MethodsCytochrome c from horse heart was obtained from Sigma Chemical Co. in the highest available grade. The lyophilized protein was dissolved in 50 mM potassium phosphate buffer in 99.8% D20 or 90% H2O/10% D20 as required. Samples used in the assignment work were reduced with 3-5 equiv of solid sodium dithionite, adjustedto pH* 5.7 with dilute NaOD or DC1 as required, and kept under nitrogen during long data acquisition periods. Protein concentration was 6-8 mM in D20 solution and 12-14 mM in H20. D20 samples were allowed to exchange with solvent in the oxidized state at pH* 6 and room temperature for 10 min prior to reduction. Two-dimensional NMR spectra were recorded on Bruker WM 500 spectrometers (Yale University, New Haven, and University of Washington, Seattle)./-Correlated (COSY) spectra were recorded with the standard pulse sequence (Aue et al., 1976; Nagayama et al., 1979; Bax & Freeman, 1981)