Kerlavage A R, Weitzmann C J, Hasan T, Cooperman B S
J Chromatogr. 1983 Aug 26;266:225-37. doi: 10.1016/s0021-9673(01)90896-9.
We have previously reported the application of reversed-phase high-performance liquid chromatography (RP-HPLC) to the separation of Escherichia coli ribosomal proteins (A. R. Kerlavage, L. Kahan and B. S. Cooperman, Anal. Biochem., 123 (1982) 342-348; A. R. Kerlavage, T. Hasan and B. S. Cooperman, J. Biol. Chem., in press). In the present studies RP-HPLC is shown to yield much greater resolution of these proteins than does size-exclusion HPLC. In addition, we report on various aspects of RP-HPLC of ribosomal proteins including column capacity, resolution, reproducibility, recovery, separation of irreversibly denatured protein, and analysis of affinity-labeled ribosomal protein. The capacity of analytical columns was found to range from several micrograms to several milligrams with minimal loss in resolution and highly reproducible retention values. Recovery varied from protein to protein and ranged from 27% to 91%, with an average total protein recovery of 70%. The partitioning of several proteins between two peaks was shown to be due to irreversible denaturation of a small fraction. Finally, the utility of RP-HPLC in the study of the ribosome was demonstrated by analyses of [3H]puromycin-labeled ribosomal proteins, and the demonstration that labeling slightly alters protein elution.
我们之前曾报道过反相高效液相色谱法（RP-HPLC）在大肠杆菌核糖体蛋白分离中的应用（A. R. 克拉瓦奇、L. 卡汉和B. S. 库珀曼，《分析生物化学》，123 (1982) 342 - 348；A. R. 克拉瓦奇、T. 哈桑和B. S. 库珀曼，《生物化学杂志》，即将发表）。在本研究中，RP-HPLC显示出比尺寸排阻HPLC能更好地分离这些蛋白质。此外，我们报道了核糖体蛋白RP-HPLC的各个方面，包括柱容量、分离度、重现性、回收率、不可逆变性蛋白的分离以及亲和标记核糖体蛋白的分析。发现分析柱的容量范围从几微克到几毫克，分离度损失最小且保留值具有高度重现性。不同蛋白质的回收率各不相同，范围从27%到91%，总蛋白平均回收率为70%。几种蛋白质在两个峰之间的分配被证明是由于一小部分蛋白质发生了不可逆变性。最后，通过对[³H]嘌呤霉素标记的核糖体蛋白的分析，以及证明标记会略微改变蛋白质洗脱情况，证实了RP-HPLC在核糖体研究中的实用性。
