Authors: Talavera, Eva M.; Guerrero, Pablo; Ocana, Francisco; Alvarez-Pez, Jose M.

Source: Applied Spectroscopy, Volume 56, Issue 3, Pages 80A-87A and 275-405 (March 2002) , pp. 362-369(8)

Publisher: Society for Applied Spectroscopy

Abstract:

High-precision time-correlated single-photon-counting data were obtained to achieve the resolution of the fluorescence decay time components from DNA-Ethidium bromide (Eb) buffered solutions at low (0.05 M) and high (0.4 M) NaCl concentrations. Tri-exponential functions are the best models to describe the decay kinetics in all solutions, independently of the NaCl concentration. The fluorescence nanosecond graphs were recorded intercalating polarizers into the light path to check for anisotropy artifacts. The results suggest that the three exponentials from fluorescence decay graphs depict three true lifetimes, which, in general, are dependent on the [DNA]/[Eb] ratio and can be assigned to free Eb and bound Eb at the low- and high-affinity DNA sites. With the normalized weighting coefficients from tri-exponential fitting of nanosecond decay graphs, recorded at the excitation wavelength of the isosbestic point, it has been possible to calculate the values of binding constants and site numbers at the two salt concentrations by means of the neighbor exclusion model (McGhee-von Hipple expression).

Keywords: TIME-RESOLVED FLUORESCENCE SPECTROSCOPY; DNA INTERCALATION BINDING CONSTANTS; ETHIDIUM BROMIDE

Document Type: Research article

DOI: http://dx.doi.org/10.1366/0003702021954737

Publication date: 2002-03-01

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