Separation and quantification of histone H1 subtypes and high-mobility-group proteins by reversed-phase liquid...

The rapid separation and quantification of histone H1 subtypes and high-mobility-group (HMG) chromatin proteins by reversed-phase liquid chromatography on a butylsilica-based column is described. The proteins were fractionated by means of a multi-step acetonitrile gradient containing 0.1% trifluoroacetic acid. This system is capable of resolving the four main HMG proteins (1, 2, 14 and 17), HMG I, protein P1 with HMG 18 and HMG 19 (in one peak) and five histone H1 subtypes in a single 33-min analysis. This method was used to study levels of these chromosomal proteins in nuclei of rat liver, spleen, testis and thymus during postnatal development from 1 to 20 weeks of age. Although no clear tissue specificity of the HMG proteins was apparent, there were significant differences in the relative amounts of these proteins in different tissues. The relative amount of HMG 1 increased from 1 to 12 weeks of age and decreased thereafter, whereas those of HMG 14 and HMG 17 remained almost unchanged. Marked quantitative differences were observed in the five histone H1 subtypes in different tissues. The largest changes in their levels during development were found in the liver and the smallest changes in the thymus. The changes in the spleen and testis were intermediate. These results suggest that the changes in the relative amounts of histone H1 subtypes and HMG proteins observed during postnatal development of the rat may result from differences in the structure of chromatin in these tissues and thus reflect the activity of molecular mechanisms involved in replication and differentiation of the cells.