Isolation and Analysis of Non-Histone Chromatin Proteins from Rat-Liver Nuclei by Three Different Methods

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Abstract
Non-histone chromatin proteins were isolated from rat-liver nuclei by three different methods, and defined as (I) phenol-soluble proteins, (II) SDS-soluble proteins and (III) proteins not adsorbed by cation-exchange chromatography. About 62–70% of chromatin proteins were recovered from the total nuclear proteins. The yield of non-histone chromatin proteins varied from 17 to 26% of chromatin proteins, depending on the method used. The amino-acid composition of these proteins showed that they are acidic in nature. Their phosphorus content was found to be 0.9, 1.1, and 1.4%, respectively, according to method I, II, or III. In-vivo pulse-labelling experiments indicated that chromatin proteins were highly labelled with 3H-acetate and 32P-phosphoric acid. In particular, the specific activities of 32P incorporation were higher in all non-histone chromatin proteins isolated as compared with histones. One-dimensional SDS–polyacrylamide gel electrophoresis showed that at least 26 similar fractions can be detected in the samples prepared by these three methods.The similarity of some of the proteins obtained from methods I and III was further confirmed by fractionation of the non-histone chromatin proteins in an isoelectro-focusing system followed by a second-dimensional SDS–polyacrylamide gel electrophoresis. It was found that more than 100 components could be identified. However, some minor variations of the non-histone chromatin proteins were detected by this system. The differences in proteins isolated by these methods are mainly quantitative rather than qualitative. The methods examined are not specific for the fractionation of a certain class of non-histone chromatin proteins.

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