ISSN:
1432-119X
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Medicine
Notes:
Summary We investigated the binding of azure B to DNA (calf thymus) over a wide range of concentrations of the dye (C F) and the nucleic acid (C N) using absorption spectroscopy [C Fand C Nrepresent the total concentrations of the dye (F) and the mononucleotide units (N) of the DNA, respectively]. The binding isotherms of the dye to DNA in aqueous solutions were determined. In addition, we analysed the composition of insoluble DNA/azure B precipitates that are formed in presence of an excess of azure B. These precipitates are of particular interest, because Giemsa staining is usually performed using high dye concentrations. Azure B easily forms dimers in aqueous solutions. When determining the binding isotherms, the equilibrium between free monomers and dimers must be taken into account. Therefore, we determined the dimerisation constant (K d) of azure B from the concentration dependency of its absorption spectra in water at the standard temperature T=298 K (25°C), K d=6.5·103 M −1 (experimental conditions: tris buffer, pH 7.2; concentration of Na⊕ ions, C Na=0.002 M). As the C Na value increases, the dimerisation constant rises rapidly. When the azure B concentration is very low and there is an excess of DNA, ordinary Scatchard and Langmuir isotherms are observed. Monomer dye cations are bound to DNA, these cations being in equilibrium with free monomers in the solution. In order to obtain the Scatchard binding constant (K S) and the binding parameter (n) spectroscopically, it is necessary to determine the extinction coefficient (ε Fb ) of the monomer bound (b) dye molecules (F) at one analytical wave number $$(\tilde v_a )$$ . The three constants can be determined simultaneously using an iterative technique that combines Scatchard isotherms and the Benesi-Hildebrand extrapolation, C N→∞. We obtained K S=1.8·105 M −1 and n=0.18 (25°C; tris buffer, pH 7.2; C Na=0.002 M). At very low dye (C F) and competitor (C Na) concentrations, only 18% of the anionic binding sites of the DNA are capable of binding the dye cations. With increasing C Na values the concentration of bound azure B cations decreases rapidly. The Na⊕ cations displace the bound dye cations and act as a competitor. The K Svalue also greatly depends on the competitor concentration (C Na). This dependency can be used to determine the binding constant of intercalation, K 1=2.7·103 M −1, the binding constant of pre-intercalative or external binding, K 2=1.9·105 M −1, and the binding constant of the competitor cations (Na⊕), K 3=24 M −1 (25°C; tris buffer, pH 7.2). The binding constant of intercalation (K 1) is very small compared with the constant of pre-intercalative binding (K 2). Therefore, at low dye and competitor concentrations, azure B behaves like a non-intercalating compound. With increasing dye concentrations (C F), pronounced positive deviations from the ordinary Scatchard and Langmuir isotherms are observed. Many more dye molecules are bound to DNA than would be expected. These positive deviations are caused by cooperative binding (q). Each anionic phosphodiester residue of DNA is a binding site for dye cations, g=1.0. Using a spectroscopic technique, we were able to determine the extinction coefficient (ε Fb ) of the cooperatively bound dye molecules at low DNA concentrations, C N→0. Using a method involving two analytical wave numbers $$(\tilde v_a , \tilde v'_a )$$ , we were able to evaluate experimentally the binding isotherm in the range of cooperative dye binding (25°C; tris buffer, pH 7.2; C Na=0.002 M). The binding isotherm was calculated by applying the theory of cooperative binding as proposed by Schwarz. Three constants characterize the binding isotherm: 1. K n=1.5·103 M −1 (the constant of nucleation); this describes the binding of a monomer dye molecule to one binding site of DNA that has unoccupied neighbour sites. 2. K q=1.2·105 M −1 (the constant of aggregation or growth); this describes the binding of a dye cation in the immediate neighbourhood of an already bound dye molecule. 3. q=K q/Kn=80 (the cooperativity parameter); this describes the interaction of the nearest neighbouring dye molecules on the DNA. It can be shown that the monomer bound azure B cations of the Scatchard or Langmuir isotherm do not represent the nucleation step of cooperative dye binding. The two binding mechanisms are different and are competitive with each other. Bearing this in mind, it was possible to calculate the isotherm over the entire concentration range of non-cooperative and cooperative dye binding. The spectra of the bound dye species could be separated from the total absorption of the solutions by using a combination of absorption spectroscopy and equilibrium dialysis. The bound monomers (M), dimers (D) and higher aggregates (A) of azure B have different absorption spectra. The first two have absorption maxima at 15 200 cm−1 (657 nm) and 17 200 cm−1 (582 nm), respectively. The higher aggregates (A) have a main peak at 18 000 cm−1 (556 nm) with two shoulders at approximately 15 300 cm−1 (654 nm) and 16 800 cm−1 (596 nm) that need to be distinguished from the absorption bands of M and D in the same spectral regions. At very low C F/CNratios, only bound monomers are observed. At somewhat higher C F/CNratios, monomers and dimers are detected. However, when the C F/CNratio increases beyond a certain point, the monomers and dimers disappear completely; instead, we observed higher aggregates of bound azure B cations exhibiting a characteristic broad absorption band at 18 000 cm−1 and two long wave-length shoulders at 15 300 and 16 800 cm−1. This behaviour is in agreement with the cooperativity parameter, q=80: Cooperative binding favours the formation of higher aggregates with increasing C F/CNratios. At relatively high dye and DNA concentrations, insoluble DNA/azure B precipitates (P) are formed. We analysed the composition of these precipitates, r P=(CFb/CN)P, as a function of C F/CN(C N=1.56·10−4 M). At a ratio C F/CN=1.8, each anionic binding site of DNA is occupied by an azure B cation, r P=1.0. The nucleic acid is saturated with dye cations, and the DNA/azure B complex is uncharged. At C F/CN〉1.8, rP〉1: dye cations are bound in excess as compared with the number of anionic binding sites of the DNA. At C F/CN=5.28 (CF=8.22·10−4 M), we found r P=1.37: The DNA/azure B complex now has a positive charge. Finally, we measured the microspectra of fine fibres or films of DNA/azure B precipitates at different C F/CNratios. In all spectra, the intense maximum of the bound higher aggregates (A) at 18 000 cm−1 (556 nm) is dominant. At relatively low C F/CNratios, the absorption of bound dimers at 17 100 cm−1 (585 nm) is also detectable. With increasing C F/CNratios, the absorption of the dimers disappears; the only remaining spectrum is that of A, with its intense maximum at 18 000 cm−1 and the two weaker bands at approximately 15 400 cm−1 (651 nm) and 16 500 cm−1 (606 nm). Again, cooperativity favours the formation of higher aggregates with growing azure B concentrations. These higher DNA-bound azure B polymers are present under the conditions prevailing during Giemsa staining. The positively charged DNA/azure B complexes are the target of eosin Y anions during the formation of the DNA/azure B/eosin Y complex, resulting in the purple colouration of the cell nuclei produced by Giemsa staining.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00533401
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