The fluorescence characteristics of ethidium bromide (Eb) complexed to calf thymus DNA have been examined using fluorescence lifetime analysis for a range of DNA (effective nucleotide concentration) to Eb molar ratios. Control of both temperature and ion concentration is necessary for reproducible analyses. Eb complexed to double stranded DNA has a maximum fluorescence lifetime of 23 ns and is easily distinguishable from a fluorescence lifetime value of 1.67 ns corresponding to unbound Eb. In a solution of calf thymus DNA containing excess EB a binding equilibrium is reached, and this corresponds to one Eb molecule for every five nucleotides. With increasing amounts of unbound Eb, the fluorescence lifetime of the DNA-Eb complex decreases with a concomitant drop in the steady state fluorescence intensity, without a change in the amount of Eb bound to DNA. It is concluded that unbound Eb, acting via a quenching mechanism, shortens the fluorescence lifetime of bound Eb and consequently decreases the overall fluorescence intensity. This means that a different approach is necessary: time-resolved fluorescence spectroscopy directly distinguishes between a decrease in fluorescence intensity due to quenching by an excess of unbound Eb from that due to a decrease in Eb binding to double-stranded DNA. These studies suggest that techniques which measure total steady state fluorescence intensity of bound Eb in order to infer relative amounts of double-stranded DNA must be interpreted with caution. For such assays to be valid it is essential that no unbound Eb be present: otherwise a variable correction factor is required to account for unbound Eb.
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http://dx.doi.org/10.1016/0301-4622(93)e0101-a | DOI Listing |
Cureus
December 2024
Medicine, College of Medicine, Taibah University, Medina, SAU.
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Institute of Biochemistry and Molecular Biology, College of Life Sciences, China Medical University, Taichung, Taiwan.
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Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata, India.
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View Article and Find Full Text PDFInorg Chem
January 2025
Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Z1, Canada.
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Department of Chemistry, Quaid-i-Azam University, Islamabad, 45320, Pakistan.
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