Studies on the Thiol Components of Isolated Nuclei

Abstract

The thiol components of the nonhistone proteins prepared from isolated nuclei from rat liver, regenerating liver and hepatoma 223 cells have been investigated after reaction with radio labelled N-ethylmaleimide and 5-5’-dithiobis-(2-nitrobenzoic acid) (DTNB). The labelled adducts formed were examined by isoelectric focusing analysis in polyacrylamide gel and the distribution of the radiolabels within sliced portions of the gels determined. In the case of the 14 C labelled NEM adduct the label was found to be spread amongst numerous protein components within the gel however, in the case of the 35 S labelled DTNB adducts, only a small proportion of the label was found in the protein material which was retained in the acidic isoelectric point (pI) region of the gel. The bulk of the 35 S labelled adduct (56% - 60%) was found to have migrated into the anode solution (10 mM phosphoric acid). This could be adsorbed onto a hydrophobic resin (XAD2) resin and eluted with methanol. Gel filtration chromatographic analysis of this adduct material on BioGel P2, (exclusion limit 1500 daltons) showed low molecular weight components to be present. Slightly different patterns were obtained for these nuclei, each containing several 35S components with molecular weights greater than the Ellman reagent itself. These 35 S labelled thiol components did not contain any protein, peptide or amino acid components indicating strongly that a novel species of thiols could be present in these nuclei bound within the non-histone protein matrices.