Ethidium bromide binding to native and denatured poly(dA)poly(dT)

Abstract

Isotherms of the EtBr adsorption on native and denatured poly(dA)poly(dT) in the temperature interval 20–70°C were obtained. The EtBr binding constants and the number of binding sites were determined. The thermodynamic parameters of the EtBr intercalation complex upon changes of solution temperature 20–48°C were calculated: 1.0·106 M−1≤K≤1.4·106 M−1, free energy ΔGo=−8.7±0.3 kcal/mol, enthalpy ΔHo≅0, and entropy ΔSo=28±0.5 cal/(mol deg). UV melting has shown that the melting temperature (Tm) of EtBr-poly(dA)poly(dT) complexes (μ=0.022,4.16·10−5 M EtBr) increased by 17°C as compared with the ΔTm of free homopolymer, whereas the half-width of the transition (Tm) is not changed. It was shown for the first time that EtBr forms complexes of two types on single-stranded regions of poly(dA)poly(dT) denatured at 70°C: strong (K1=1.7·105 M−1; ΔGo=−8.10±0.03 kcal/mol) and weak (K2=2.9·103 M−1; ΔGo=−6.0±0.3 kcal/mol).The ΔGo of the strong and weak complexes was independent of the solution ionic strength, 0.0022≤μ≤0.022. A model of EtBr binding with single-stranded regions of poly(dA)poly(dT) is discussed.