Search

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)

A reproducible Romanowsky-Giemsa staining (RGS) can be carried out with standardized staining solutions containing the two dyes azure B (AB) and eosin Y (EY). After staining, cell nuclei have a purple coloration generated by DNA-AB-EY complexes. The microspectra of cell nuclei have a sharp and intense absorption band at 18,100 cm-1 (552 nm), the so called Romanowsky band (RB), which is due to the EY chromophore of the dye complexes. Other absorption bands can be assigned to the DNA-bound AB cations. Artificial DNA-AB-EY complexes can be prepared outside the cell by subsequent staining of DNA with AB and EY. In the first step of our staining experiments we prepared thin films of blue DNA-AB complexes on microslides with 1:1 composition: each anionic phosphodiester residue of the nucleic acid was occupied by one AB cation. Microspectrophotometric investigations of the dye preparations demonstrated that, besides monomers and dimers, mainly higher AB aggregates are bound to DNA by electrostatic and hydrophobic interactions. These DNA-AB complexes are insoluble in water. Therefore it was possible to stain the DNA-AB films with aqueous EY solutions and also to prepare insoluble DNA-AB-EY films in the second step of the staining experiments. After the reaction with EY, thin sites within the dye preparations were purple. The microspectra of the purple spots show a strong Romanowsky band at 18,100 cm-1. Using a special technique it was possible to estimate the composition of the purple dye complexes. The ratio of the two dyes was approximately EY:AB approximately 1:3. The EY anions are mainly bound by hydrophobic interaction to the AB framework of the electrical neutral DNA-AB complexes. The EY absorption is red shifted by the interaction of EY with the AB framework of DNA-AB-EY. We suppose that this red shift is caused by a dielectric polarization of the bound EY dianions. The DNA chains in the DNA-AB complexes can mechanically be aligned in a preferred direction k. Highly oriented dye complexes prepared on microslides were birefringent and dichroic. The orientation is maintained during subsequent staining with aqueous EY solutions. In this way we also prepared highly orientated purple DNA-AB-EY complexes on microslides. The light absorption of both types of dye complexes was studied by means of a microspectrophotometer equipped with a polarizer and an analyser. The sites of best orientation within the dye preparations were selected under crossed nicols according to the quality of birefringence.(ABSTRACT TRUNCATED AT 400 WORDS)

We investigated the binding of azure B to DNA (calf thymus) over a wide range of concentrations of the dye (CF) and the nucleic acid (CN) using absorption spectroscopy [CF and CN represent the total concentrations of the ye (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 (Kd) of azure B from the concentration dependency of its absorption spectra in water at the standard temperature T = 298 K (25 degrees C), Kd = 6.5 X 10(3) M-1 (experimental conditions: tris buffer, pH 7.2; concentration of Na ions, CNa = 0.002 M). As the CNa 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 (Ks) and the binding parameter (n) spectroscopically, it is necessary to determine the extinction coefficient (epsilon Fb) of the monomer bound (b) dye molecules (F) at one analytical wave number (upsilon a). The three constants can be determined simultaneously using an iterative technique that combines Scatchard isotherms and the Benesi-Hildebrand extrapolation, CN----infinity. We obtained Ks = 1.8 X 10(5) M-1 and n = 0.18 (25 degrees C; tris buffer, pH 7.2; CNa = 0.002 M). At very low dye (CF) and competitor (CNa) concentrations, only 18% of the anionic binding sites of the DNA are capable of binding the dye cations. With increasing CNa values the concentration of bound azure B cations decreases rapidly. The Na cations displace the bound dye cations and act as a competitor. The Ks value also greatly depends on the competitor concentration (CNa).(ABSTRACT TRUNCATED AT 400 WORDS)

The fluorescent staining of fixed A9 cells (mouse fibroblasts) with ethidium bromide E has been investigated qualitatively and quantitatively. E is bound to DNA, RNA and protein. After enzymatic RNA digestion and staining at pH = 4.0 the dyestuff is bound to DNA specifically. The formed E-DNA-complex is stoichiometric if DNA will be saturated by highdye concentration, CF = 1.0 x 10(-2) M. The stoichiometric factor n = 0.21 was determined from Scatchard binding isotherms and by spectrophotometric titration. The binding of E to DNA and RNA has been investigated by binding isotherms. E is bound to DNA by intercalation and by ionic interaction. It is bound to RNA by ionic interaction only. The ionically bound dyestuff can be replaced by salts like LiCl from DNA and RNA. Only intercalated E remains. The competitive salt effect can be used to avoid the enzymatic RNA digestion in the specific staining of DNA. Within the cell the E-DNA-complex fluoresces strongly. Using a microspectrophotometer we determined the fluorescence intensity J and the extinction E in cell sections of q = 1 mu 2 area. The fluorescence was excited in the minimum of the absorbance of the E-DNA complex at lambda 1 = 365 nm. The extinction E2 was measured in the short wavelength band lambda 2 = 260 nm. Under these conditions we received a linear relation J = J (E2) up to high extinctions E2. The quantum yield Q of the E-DNA-complex is nearly constant and independent of the biological environment. Q is also unaltered by LiCl. Within the limits of error intercalated and ionic bound dye have the same quantum yeild. Under the conditions of stoichiometric staining the amount of E-DNA-complex and therefore DNA in the cell can be determined by J. The extinction coefficient epsilon 2 = 45 200 M-1 cm-1 of the E-DNA-complex at 260 nm was calculated from the concentration of dependence of the absorbance spectra using the law of mass action. By epsilon 2 the amount of DNA in the cell is accessible from J. According to our measurements with A9 cells the superficial density of DNA has the order of magnitude rho N = mN/q = 10(1) nmol cm-2, the amount mN of DNA per section 1 = 1 mu 2, mN = 10(-1) fmol, and the DNA concentration in the cell nucleus CN = 10(-1) M. The ray pass and diagram of electronics of the based microspectrophotometer are described.