Stopped-flow kinetic, 1H, and 31P NMR analysis of the intercalation of ethidium with poly d(G-C) X d(G-C)

In a low salt buffer (0.011 M Na+) stopped-flow kinetic results for the SDS driven dissociation of an ethidium-Poly d(G-C) X d(G-C) complex are 8.7, 23, and 58.5 s-1 at 20, 30, and 40 degrees C, respectively. These results predict that in NMR experiments at high field strengths, ethidium should be in slow exchange among polymer binding sites. This has been found to be the case for both 31P (109 MHz) and 1H (imino proton spectra in H2O at 270 MHz) experiments. At higher salt, and/or higher temperature, and/or lower field, the bound and free peaks are no longer resolved in the NMR spectra. Good agreement is obtained between the stopped-flow kinetic results and the coalescence temperature observed in NMR experiments. Imino protons in base pairs on both sides of the intercalated ethidium are shifted approximately one ppm upfield while only the phosphate groups at the intercalation site experience large chemical shifts.