HETEROGENEITY OF ETHIDIUM-BROMIDE STRONG BINDING-SITES ON DNA - FLUORESCENT AND NONFLUORESCENT COMPLEXES

The isotherms of ethidium bromide (EtBr) adsorption on DNA of differen t base composition were studied over a range of solution ionic strengt h (0.01 less than or equal to [Na+] less than or equal to 0.5 M) and t emperature (20 less than or equal to t less than or equal to 40 degree s C). The fluorescent and nonfluorescent strong complexes on DNA were registered by absorption and fluorimetry. The binding constant (K) and stoichiometry (n) (the amount of base pairs (bp) necessary to bind on e ligand molecule) of the first (K-f, n(f)) and second (K-p, n(p)) com plexes were calculated. K-f changed from 5 . 10(6) to 10(5) M-1. K-p r emained lower than K-f (K-p/K-f less than or equal to 0.3). The n(f) v alue equal to 2 bp was independent of the solution ionic strength and GC content in DNA, whereas n(p) changed from 18 bp to 5 bp upon increa sing the GC portion from 0 to 50%. Thermodynamic parameters of EtBr-DN A complexes were calculated. The fluorescent complex was more stable t han the nonfluorescent one. The fluorescent complex is formed upon EtB r intercalation in the double helix. The nonfluorescent complex meets the requirements of the small groove model for DNA with an increased G C content.