Ethidium ion binds more strongly to a DNA double helix with a bulged cytosine than to a regular double helix.

Thermodynamic parameters for ethidium intercalation were determined for the double helices formed by the oligonucleotides dCA6G + dCT6G, which form a normal helix, and dCA3CA3G + dCT6G, which form a double helix with the middle cytosine bulged outside of the helix. Ethidium intercalation was measured by monitoring the absorbance at 260 and 283 nm as a function of temperature for a number of concentrations of ethidium. The binding to the normal helix occurs equally at all the intercalation sites, with an enthalpy of binding of -8 kcal mol-1, an entropy of binding of -6 eu, and an equilibrium constant at 25 degrees C of 2.2 X 10(4) M-1. The binding to the bulged double helix was considerably stronger and is consistent with a model in which the intercalation sites on either side of the bulged base bind 10 times stronger than the other sites. Thus, there are two strong binding sites on the perturbed helix with equilibrium constants for binding of 2 X 10(5) M-1 at 25 degrees C in addition to five normal sites. Several other binding models were tested but did not fit the data satisfactorily.