Comparison of rat liver and calf thymus histone fractions by molecular exclusion chromatography and ultracentrifugation

Abstract

The total histone fraction of rat liver nuclei isolated in 0.44 m sucrose at two different pH values has been studied by column chromatography using Sephadex G-200, by ultracentrifugation, by amino acid analysis, and by disc gel electrophoresis. Subfractions have also been investigated. The results have been compared with comparable results obtained from calf thymus histone. Three principal peaks were observed in Sephadex chromatography which corresponded to material of average molecular weights of approximately 445,000 (peak A), 47,000 (peak B), and 300 (peak C). Sedimentation constants were used in conjunction with the results of chromatography in determining the molecular weights of peaks A and B. Whole histone extract exhibited two ultracentrifuge peaks, the slower of which corresponded to the bulk of the histone. Disc gel electrophoresis of whole histone extracts from nuclei isolated in 0.44 m sucrose at pH 3.8 and pH 5.8, and in 0.44 m sucrose-0.005 CaCl 2 without pH control showed several heavily staining bands which corresponded to the bulk of protein in the histone extract. Several lightly staining bands were also observed when 5.24 m urea was present in the gels. These presumably corresponded to impurities carried over from the “globulin” fraction. Finally, one or two lightly staining bonds were also observed in some cases. Disc gel electrophoresis was also used to show that the material corresponding to Sephadex peak B and C corresponded mainly to histone or (in the case of peak C) degraded histone. Insufficient material was collected to prove the presence of histone in peak A. Amino acid analysis showed that the material from Sephadex peak A contained most of the phenylalanine and tyrosine of the total histone extract. By comparing results obtained from Sephadex chromatography and disc gel electrophoresis using rat liver nuclei isolated in 0.44 m sucrose at pH 3.8, 0.44 m sucrose at pH 5.8, or in some cases 0.44 m sucrose-0.005 m CaCl 2 without pH control, it was found that substantial quantitative differences occurred which apparently were caused mainly by differences in the degree of histone degradation during isolation of the nuclei by different procedures.