Equilibrium binding of DAPI and 7-aminoactinomycin D to chromatin of cultured cells

Abstract

The binding of 4',6-diamidino-2-phenylindole (DAPI) and 7-aminoactinomycin D (7-AAMD) to chromatin of intact cultured fibroblast nuclei was studied by cytofluorometry. Staining was performed at equilibrium at varying dye concentrations. By using large volumes of dye solutions, the free dye concentration became approximately equal to total dye concentration, and the estimation of affinity and number of binding sites was performed by Scatchard analysis. Since all Scatchard plots showed a definite curvature, indicating more than one class of binding sites, a two component regression was performed, although only the highest affinity binding sites were determined. The observed affinity constants did not change substantially between different preparational procedures for any of the dyes. The number of DAPI binding sites increased about 30% after detergent extraction. No such increase was observed for 7-AAMD. Fixation in formaldehyde, instead of ethanol, after detergent extraction had no effect on DAPI binding, but decreased the number of 7-AAMD binding sites with about 50%. Extraction of basic proteins with HCl resulted in about 100% increase of the number of binding sites for both dyes. It should be feasible to use this technique as an assay for the identification of proteins or other nuclear components important for the maintenance of chromatin structure.