High-performance liquid chromatography of chromatin histones

A method is described for the rapid analysis of histones by high-performance liquid chromatography on reversed-phase muBondapak columns, containing either octadecylsilane (C18) or cyanopropylsilane (CN) bonded to silica particles packed in either steel columns or Radial-Pak cartridges. A linear gradient progressing from water-acetonitrile (80:20) to water-acetonitrile (40:60) and increasing in acetonitrile concentration at the rate of 10%/h was used to elute the histones at a flow-rate of 1 ml/min for steel columns or 2 ml/min for Radial-Pak cartridges. Two conditions were found to be necessary to achieve histone fractionation: (1) silylation of the active silanol groups on the silica matrix, and (2) 0.1-0.3% trifluoroacetic acid in the eluting solvent. More than 95% of the total [3H]lysine-labeled protein applied to the CN column was eluted. The histone fractions were identified by their electrophoretic mobilities in both acid-urea and Triton DF-16 polyacrylamide gels. Histones were eluted from the columns in the following order: H1, H2B, (LHP)H2A, (MHP)H2A, H4, (LHP)H3, and (MHP)H3 (where LHP and MHP refer to the less-hydrophobic and more-hydrophobic histone variants). Phosphorylated and acetylated histone molecules were not separated from their unmodified parent molecules. The volatile nature of the water-acetonitrile-trifluoroacetic acid eluting solvent facilitated recovery of salt-free histones from the fractions by direct lyophilization of the column effluents. The best resolution of histone fractions was obtained with the Radial-Pak muBondapak C18 cartridge using 0.3% TFA. However, for analytical studies, the best detection was obtained by using the muBondapak CN steel column. Poorer resolution was obtained by using the non-silica based PRP-1 reversed-phase column, containing a polystyrene-divinylbenzene resin under the same conditions.