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Binding of ethidium to bacteriophage T7 and T7 deletion mutants

1985; Wiley; Volume: 24; Issue: 8 Linguagem: Inglês

10.1002/bip.360240816

1097-0282

Gary A. Griess, Philip Serwer, Paul M. Horowitz,

Advanced biosensing and bioanalysis techniques

Abstract Equilibrium binding of ethidium, quantitated by fluorescence enhancement, to DNA packaged in bacteriophage T7 and T7 deletion mutants has been compared with the binding of this dye to DNA released from its capsid (free DNA). During achievement of apparent equilibrium binding, no change in bacteriophage T7 structure occurred, by the criterion of agarose gel electrophoresis. However, excessive incubation with ethidium bromide caused detectable changes in bacteriophage structure, a possible explanation of disagreements in similar studies previously performed with T‐even bacteriophages. Scatchard plots for packaged DNA had a curvature greater than the previously demonstrated [Bresloff, J. L. & Crothers, D. M. (1981) Biochemistry 20 , 3547–3553] curvature for free DNA. By treating plots for packaged DNA as though they were biphasic, it was found that binding to most sites occurred with an apparent association constant ( K ap ) 3.3–4.3 times lower than the K ap of free DNA. The number of these sites increased significantly as the density of packaged DNA was decreased by use of the deletion mutants. Values of Δ H ° for these sites were negative and equal to the Δ H ° for free DNA; values of Δ S ° were positive and about half the Δ S ° for free DNA. A second class of sites, roughly 1.2% of the total, had a significantly higher K ap and more negative Δ H ° than those of the majority of sites.