Separation of phosphorylated histone H1 variants by high-performance capillary electrophoresis.

High-performance capillary electrophoresis (HPCE) was used to separate successfully distinct phosphorylated derivatives of individual histone H1 variants. With an untreated capillary (50 cm x 75 microns I.D.) the electrophoresis was performed in about 15 min. Inconvenient interactions of these highly basic proteins with the capillary wall were eliminated by using 0.1 M sodium phosphate buffer (pH 2.0) containing 0.03% hydroxypropylmethylcellulose. Under these experimental conditions the histone H1 variants H1b and H1c obtained from mitotic enriched NIH 3T3 fibroblasts and isolated by reversed-phase high-performance liquid chromatography were clearly separated in their non-phosphorylated and different phosphorylated forms. This result was confirmed by acid-urea gel electrophoresis, comparison with non-phosphorylated histones H1b and H1c, isolated from quiescent NIH 3T3 cells, and incubation of multi-phosphorylated histone H1b with alkaline phosphatase and subsequent acid-urea and capillary electrophoresis. The results illustrate that the application of HPCE to the analysis of histone modifications provides a new alternative to traditional gel electrophoresis.

High-performance capillary electrophoresis of core histones and their acetylated modified derivatives.

By using high-performance capillary electrophoresis, we have successfully separated rat liver core histones into several subfractions. Inconvenient interactions of the highly basic proteins with the capillary wall were eliminated by a phosphate buffer system containing 0.03% hydroxyprophylmethylcellulose. Sample amounts of a few nanolitres were analysed within about 20 min. Multiacetylated histones H4 and H3 from induced Friend erythroleukaemic cells prepurified by h.p.l.c. were clearly separated into their non-acetylated and distinct acetylated forms. Our results illustrate that the application of capillary zone electrophoresis on its own or in combination with h.p.l.c. to the analysis of histones provides an important new alternative to traditional gel electrophoreses.