Open AccessBinding of Ethidium Bromide to Self‐Complementary Deoxydinucleotides
Author(s) -
DOGLIA Silvia,
GRÄSLUND Astrid,
EHRENBERG Anders
Publication year - 1983
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1983.tb07445.x
Subject(s) - chemistry , titration , ethidium bromide , bromide , fluorescence , absorption spectroscopy , absorption (acoustics) , equilibrium constant , binding constant , analytical chemistry (journal) , spectral line , titration curve , binding site , inorganic chemistry , chromatography , materials science , dna , biochemistry , physics , quantum mechanics , astronomy , composite material
Optical spectra titrations were performed with ethidium bromide and the self‐complementary deoxydinuclectides pdCpdG, pdGodA, pdTpdA, and pdApdT. The titrations were performed in 7.5 mM phosphate buffer, pH 7.0, at 7°C, and with varying dinucleotide concentrations always in large excess of the dye concentration. Well‐defined isosbestic points were present in each titration after correction for dinucleotide light absorption. The binding curves were evaluated in terms of simple bimolecular or termolecular reaction models. The bimolecular reaction model gave a significantly better fit to the experimental data, judging from a computerized non‐linear least‐squares fitting procedure. The following equilibrium constats were obtained: K C‐G = 2000 M −1 ; K G‐C = 950 M −1 ; K T‐A = 370 M −1 ; K A‐T = 350 M −1 . From these data the absorption spectra of the completely bound dye were evaluated. These spectra showed bathchromic shifts of their maxima, increasing with the magnitude of K . Fluorescence spectra of ethidium bromide/dinucleotide mixtures were recorded under conditions similar to those for absorption spectra. From the known equilibrium constants the contributions of the bound dye could be estimated. The following fluorescence enhancements I b / I f wer found: I b C‐G / I f = 6.5: I b G‐C / I f = 3.0; I b T‐A / I f = 2.0; I f A‐T / I f = 2.0. From our results, in relation to other theoretical and experimental studies, we conclude that electrostatic phosphate‐dye interactions give rise to a major part of the binding energy, which varies with dinucleotide geometry. The more strongly bound complexes exhibit less exposure of the dye to the solvent.