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1. 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.2. I n 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.3. 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. 4. 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 ionexchange material is not entirely dependent on the overall charge of the polypeptide. 5. Preparation of individual nonhistone protein species has been attempted using this chromatographic procedure in conjunction with gel filtration in guanidine hydrochloride.The activities which regulate the process of transcription in eukaryotes appear to reside in the nonhistone protein fraction rather than amongst the histones of the chromatin (see review 111-1). I n particular, nonhistone proteins have been implicated in the activation ofthe genome by hormones in mammals [2] and in insect larvae [3], in altering the transcription of both DNA [4,5] and chromatin [6,7] by bacterial RNA polymerase in vitro and in the tissuespecific restriction of the template capacity of DNA in chromatin [S-lo]. A significant proportion of the nonhistone proteins are phosphoproteins [ 111 whose phosphate groups are rapidly labelled [12] and in addition a number of workers [a, 13,141 have shown that some of the effects on transcription described above are associated with such modified proteins.Abbreviation. QAE-Sephadex, quaternary diethyl-(2-hydroxypropy1)-aminoethyl-Sephadex.

The effect of the dissociation medium on the fractionation of chromatin on hydroxyapatite has been studied. Optimal separations of the histones and non-histone protein are only achieved when columns are run in buffers containing high concentrations of sodium ions. We have modified our previously published method such that the chromosomal proteins can be recovered in virtually quantitative yields. Each of the hydroxyapatite fractions has been analysed with respect to nucleic acid content and the proteins have been analysed by two-dimensional gel electrophoresis.Recent evidence suggests that the non-histone proteins of chromatin play an important role in the control of transcription (see review by MacGillivray and Rickwood [l] [6] after the chromatin has been extracted with dilute acid to remove the basic histones. Alternatively, chromatin has been dissociated in sodium dodecyl sulphate [7] or in various salt solutions [8-201 so that after removal of the DNA by centrifugation [7][8][9]12,13,16,17] gel filtration [9-11,18, 191 or precipitation [14,15,20], the chromatin proteins have been obtained as such [I41 or separated from the histones by ion-exchange chromatography [8,[10][11][12][13][15][16][17][18][19][20] or by electrophoresis [7]. Other workers have used variations or combinations of some of these techniques [21-241. However, some of these methods subject the chromatin to extremes of pH and solvent environment which can be destructive to both proteins [25] and DNA [5,19], while others produce selected fractions [2,3,15] or yield low recoveries of non-histone proteins [ l l , 16,18,19]. Our approach has been to use non-rigorous conditions which yet give good yields of non-histone proteins. In our procedure chromatin is dissociated in 2 M NaCl-5 M urea and applied to a column of hydroxyapatite. This ion-exchange medium is not affected by the presence of high concentrations of urea and the phosphate sites which would otherwise bind basic molecules such as histones are effectively blocked by the sodium ions [26,27]. The acidic proteins bind to the Ca2+ sites of the column and are eluted by increasing the phosphate concentration [26]. Thus in the case of chromatin, we found that the histones are not retained and can be recovered in almost quantitative yield, but only 60%-70% of the adsorbed nonhistone proteins could be recovered using phosphate concentrations up to 0.2 M [28,29]. Further increase in the phosphate concentration merely eluted the DNA from the hydroxyapatite. Recently we have found that solutions of high concentrations of guanidinium hydrochloride to be an efficient solvent for non-histone proteins [30,31]. This communication describes the adaption of our previous procedures to include the use of guanidinium hydrochloride in order to obtain a more efficient recovery of nonhistone proteins. MATERIALS AND METHODS ChemicalsAs far as possible these were of AnalaR grade and were largely obtained from British Drug Houses Ltd (Poole, Dorset, U.K.). AnalaR-grade urea was routinely prepared as an 8 M sto...

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