Reinvestigation of the binding of proflavine to DNA. Is intercalation the dominant binding effect?
The binding isotherms of the acridine dye proflavine (PF) to calf-thymus DNA have been carefully redetermined at pH = 6.5 and 25 degrees C. Bound monomers (M), dimers and trimers (D,T) as well as higher polymeric PF cations (P) could be distinguished by absorption spectroscopy. The concentrations of these species were determined as a function of the concentration CMf of free PF cations in solution. NaCl is a competitor (S) for the bound dye cations. The competitive salt effect has been carefully studied in the wide concentration range 0 less than or equal to Cs less than or equal to 1 M. - At very low dye concentrations CMf monomeric and dimeric PF cations M and D are bound to DNA. The binding constant KM of the monomers M depends strongly on the concentration Cs of the competitor and decreases with increasing Cs to a limiting value greater zero. We have developed an extended equilibrium model for the quantitative description of this competitive salt effect. We assume that two types 1 and 2 of the bound monomers exist already at very low CMf and that they can be distinguished by the competitive salt effect: The intercalated monomers 1 are resistant to competitors whereas the pre-intercalatively bound monomers 2 are displaced by Na+ cations. The binding constants of 1 and 2 have been determined by the use of the equilibrium model: KM1 = 3.5 x 10(4) M-1, KM2* = 2.7 x 10(6) M-1. Thus, the binding constant KM1 of the intercalation 1 is very much smaller than the binding constant KM2* of the pre-intercalative bond 2. This is in contradiction to the generally accepted notion that intercalation should be the dominant binding effect. The concentration of bound monomers decreases rapidly with increasing CMf. Instead, DNA-bound dimers D and subsequently trimers T are observed. Both species are very sensitive to competitor cations. We have determined the dependence of the concentrations of bound D and T on the competitor concentration Cs using the extended equilibrium model. The respective binding constants are KD* = 8.7 x 10(4) M-1 and KT* = 2.4 x 10(4) M-1. Finally D and T disappear with increasing CMf. At higher dye concentrations, higher dye polymers P are observed instead of D and T. In contrast to D and T, the binding of P is favoured by cooperative effects.(ABSTRACT TRUNCATED AT 400 WORDS)
Schelhorn
T
T
Institut für Physikalische Chemie, Universität Freiburg, Federal Republic of Germany.
Kretz
S
S
Zimmermann
H W
HW
D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D004247:DNA / Q000378:metabolism*; D003956:Dialysis; D007364:Intercalating Agents / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D011370:Proflavine / Q000378:metabolism*; D012965:Sodium Chloride / Q000378:metabolism; D013053:Spectrophotometry; D013816:Thermodynamics
D007364:Intercalating Agents; D012965:Sodium Chloride; D004247:DNA; D011370:Proflavine