Bindungskonstanten, Bindungsenthalpien und Entropien für die nicht-kompetitive und die kompetitive Bindung von Acriflavin, Tetramethylacriflavin und Acridinorange an DNA / Binding Constants, Binding Enthalpies and Entropies of the Non-Competitive and the Competitive Binding of Acriflavine, Tetramethylacriflavine and Acridine Orange to DNA
Authors: Wille Harald , Pauluhn Jürgen , Zimmermann Herbert W. 
Published: August 2018

The binding of the dye cations acriflavine AF, tetramethylacriflavine TMAF and acridine orange AO (scheme of structures) to calf thymus DNA has been investigated by means of absorption spectroscopy, Table I. In order to avoid dye association we used very low dye concentrations and sufficiently high DNA concentrations. In this case we got linear Scatchard isotherms. The formal Scatchard binding constant K strongly depends on the salt concentration Cs (S = NaCl) of the solution and the temperature T (278 - 303 K), K (CS, T). The average value of binding sites per mononucleotide is n = 0.17. It is independent of the dye species and of CS and T.

The value of r (bound dye cations per mononucleotide) diminishes with growing salt concentration CS(CS ≲ 1 ᴍ). At sufficiently high salt concentrations r is approximately constant (CS ≳ 1 ᴍ). Obviously there are two types of binding of the dye cations to DNA even in the domain of linear Scatchard isotherms. They can be distinguished experimentally with the competitive salt effect. To describe r(CS,T ) or K (CS, T) we used a simple model with three equilibria: 1. Noncompetitive binding 1 (intercalation) of dye cations to n1 CN binding sites (CN = concentration of mononucleotides), equilibrium constant K1 . 2. Competitive binding 2 (external binding) of dye cations to n2 CN binding sites, equilibrium constant K2. In contrast to type 1 binding, the dye cations in type 2 binding can be replaced by metal cations M of S (M = Na⊕) at sufficiently high salt concentrations CS. 3. Competitive binding 3 of M to the same sites of 2 and the dye cations as competitor, equilibrium constant K3. The model agrees very well with the experiments on the condition n1 = n2 = n. Therefore the dye can be bound to one of the n CN binding sites either non-competitively or competitively. Type 1 and type 2 binding exclude one another at the same binding site in the domain of linear Scatchard plots.

The binding constants Ki(i = 1, 2, 3) have been determined by means of the competitive salt effect, Table II. They only are T dependent. From K(T)i we got the binding enthalpies ΔHi
0 and binding entropies ΔSi
0, Table III.

AF and AO cations are bound non-competitively and competitively, TMAF only competitively. In comparison with AF or AO the competitive binding of TMAF is much weaker. In the case of AF and AO K1 is approximately one power of ten smaller than K2, K1 ⪡ K2 ! The binding enthalpies of the non-competitive and the competitive binding are nearly equal, ΔH1
0 ≅ ΔH2
0. Therefore the difference in the binding constants K1, K2 can be attributed to the difference in the binding entropies, ΔS1
0 ⪡ ΔS2
0. Thermodynamically type 2 binding (external binding) is preferred to type 1 binding (intercalation).

The binding enthalpy of Na⊕ to DNA is in all cases nearly zero, ΔH3
0 ≅ 0. Only the increase of entropy S3
0 > 0 enables binding 3.

From the thermodynamic data follows that type 1 and type 2 binding of AF and AO are produced by electrostatic and hydrophobic interaction which are intensified by hydrogen bonding. In contrast to this the weaker competitive binding of TMAF is caused by electrostatic and hydrophobic interaction only. Our investigations agree with former works on ethidium bromide E and tetramethylethidium bromide TME (scheme of structures, Tables II and III). They are consistent with the assignment non-competitive binding = intercalation, competitive binding = external binding.