Histone separation by high-performance liquid chromatography on C4 reverse-phase columns.

Previous work in our laboratory (Lindner, H., Helliger, W., and Puschendorf, B. (1986) J. Chromatogr. 357, 301-310) described a rapid separation of H1 and core histones by reverse-phase high-performance liquid chromatography using a Bio-Rad Hi-Pore butyl (C4) silica-based column. Despite the short elution time, a high resolution of the different histone fractions, except H4 and H2A (MHP), could be obtained. In this report we present a method for the separation of H4 and H2A (MHP) as well, while maintaining a similar analysis time. By varying the gradient, trifluoroacetic acid concentration (0.05%), and flow rate (1.3 ml/min) the histones were eluted from the C4 column in the following order: H1 (MHP), H1 (LHP), H2B, H2A (LHP), H4, H2A (MHP), H3 (LHP), and H3 (MHP). LHP and MHP refer to less and more hydrophobic histone variants. The identification of the individual protein fractions was performed by comparison the retention times with pure histone markers as well as by gel electrophoresis.