RESUMO
The fluorochrome AMHA (3-amino-6-methoxy-9-(2-hydroxyethylamino)acridine) stains the nuclear chromatin and the chromosomes of living HeLa cells. At relatively low dye concentrations CF less than or equal to 10(-4) M and short incubation periods tI less than or equal to 2 h cell growth is not affected by the drug. But at higher CF and longer tI the population doubling time of the cell cultures rapidly increases, and finally the cells die. In vital staining experiments the dye AMHA preferentially binds to the DNA of the nuclei and to the chromosomes of the cells, respectively. The dye binding to DNA has been proved by the absorption and emission microspectra of the stained cells, and by the comparison with authentic spectra of AMHA bound to DNA in aqueous solutions. Within the limits of experimental errors both types of spectra are identical. The spectra of DNA-bound AMHA show a characteristic gap of ca. 3500 cm-1 between the 0-0-transitions of the long wave length 1La absorption and the fluorescence. AMHA molecules dissolved in the polar solvent water have a gap of even 4100 cm-1. This energy gap shows that the electron distribution of AMHA is strongly changed by light absorption and emission. Finally, using absorption spectroscopy, we investigated the binding of AMHA to DNA in aqueous solutions over a wide range of concentrations of the dye, of nucleic acid (calf thymus), and of the competitor NaCl respectively. The Scatchard binding isotherms were determined. With the method of competitive salt effect three different bonds of AMHA to DNA can be distinguished even at low dye concentrations: The intercalation 1 of the fluorochrome F, binding constant KF1 = 1.1.10(5) M-1, binding parameter n1 = 0.15; the pre-intercalative or external binding 2, KF2 = 6.9.10(5) M-1, n2 = 0.21; the external binding 3, KF3 = 2.8.10(5) M-1, n3 = 0.55. Externally bound dye molecules 2 and 3 occupy two phosphodiester residues of the DNA. A detailed discussion of the data and the competitive salt effect shows that in living cells only intercalated and small amounts of pre-intercalatively bound molecules 1 and 2 exist. The binding constant KF1 = 1.1.10(5) M-1 of AMHA is unusual high in comparison with the constants of intercalation of other dyes, KF1 = (1-4).10(4) M-1.(ABSTRACT TRUNCATED AT 400 WORDS)
