The Heterogeneity of Mouse‐Chromatin Nonhistone Proteins as Evidenced by Two‐Dimensional Polyacrylamide‐Gel Electrophoresis and Ion‐Exchange Chromatography

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Abstract
A two‐dimensional gel electrophoresis procedure employing a combination of isoelectric focusing and dodecylsulfate electrophoresis has been used to analyse the components of 32P‐labelled nonhistone protein fractions obtained by the chromatography of salt‐urea dissociated chromatins on hydroxyapatite. In this way the nonhistone proteins eluted from hydroxyapatite by 0.05 M phosphate (fraction H2) have been found to consist of a heterogeneous mixture of components of a molecular weight range of 15000 to 200000 and to have isoelectric points between pH 4.5 and 9. The components of the fraction eluted from hydroxyapatite by 0.2 M phosphate (fraction H3) appeared to consist of a smaller group of proteins with a molecular weight range similar to that of the H2 proteins but whose isoelectric points lay between pH 2 and 6. Both fractions consisted of a mixture of phoshorylated and nonphosphorylated protein species. Comparison of the components of H2 and H3 protein fractions by this electrophoretic procedure showed that many of the phosphorylated and nonphosphorylated proteins were common to mouse liver, kidney and brain chromatins. Only a few species were found to be tissue specific. It was found that chromatography on QAE‐Sephadex did not follow the same separation pattern as that obtained by isoelectric focusing, suggesting that the fractionation on this ion‐exchange material is not entirely dependent on the overall charge of the polypeptide. Preparation of individual nonhistone protein species has been attempted using this chromatographic procedure in conjunction with gel filtration in guanidine hydrochloride.