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Viscosity dependence of ethidium-DNA intercalation kinetics
R B Macgregor1, R M Clegg, T M Jovin
1Abteilung Molekulare Biologie, Max-Planck-Institut für biophysikalische Chemie, D-3400 Göttingen, Federal Republic of Germany.
Biochemistry
|June 30, 1987
Summary
Ethidium intercalation into DNA is slowed by organic solvents, suggesting a DNA conformational change is required before binding. This DNA opening step is faster than intercalation itself.
Area of Science:
- Biophysical Chemistry
- Molecular Biology
- Drug-DNA Interactions
Background:
- Ethidium bromide is a fluorescent intercalating agent used to detect nucleic acids.
- Understanding the kinetics of drug-DNA intercalation is crucial for drug design and molecular diagnostics.
Purpose of the Study:
- To investigate the kinetic mechanisms of ethidium intercalation into double-stranded poly[d(G-C)] DNA.
- To elucidate the role of solvent properties on the intercalation process.
Main Methods:
- Repetitive pressure-jump chemical relaxation experiments were conducted at 20°C.
- Experiments utilized low ionic strength aqueous buffers with varying concentrations of glycerol or methanol.
- Kinetic relaxation times were measured as a function of DNA concentration and solvent composition.
Main Results:
- The association rate constant for ethidium intercalation decreased in organic-aqueous mixtures compared to water.
- The association rate constant was inversely proportional to solvent viscosity, indicating a viscosity-dependent step.
- Evidence suggests an obligatory DNA conformational change precedes ethidium intercalation, with an equilibrium constant favoring the non-intercalating state.
Conclusions:
- An obligatory DNA conformational change, likely DNA opening, is a prerequisite for ethidium intercalation.
- The rate of this conformational change is faster than the intercalation reaction, with the reverse rate exceeding approximately 500 s⁻¹.
- Solvent density influences ethidium dissociation, suggesting a dependence on solvent free volume.