Quantitative studies on the interaction of azure a with deoxyribonucleic acid and deoxyribonucleoprotein

Abstract

The interaction of the cationic dye azure A with DNA or deoxyribonucleoprotein can result in two interdependent phenomena: at relatively low concentrations of the dye (< 2·10 −5 M) or with excess of the polyanion, a metachromatic complex appears in the solution, whereas at higher dye concentrations and with excess of dye the polyanion-dye complex precipitates. In the latter case, the amount of dye bound in the precipitate can be calculated from measuring the absorbancy of the supernatant free dye left in the dye bath. Utilizing this principle, the dyebinding of DNA and DNPr has been determined quantitatively and various conditions which influence the extent of the binding, such as pH, the presence of salts and the effect of dye concentration, have been investigated for both substances. The results indicate a quantitative, stoichiometric binding of azure A by DNA, corresponding to a 1:1 molar ratio between DNA phosphate and the dye bound. In the case of DNPr, the corresponding ratio is about 1:2, indicating that only about half of the binding sites is available for the dye. Whereas the binding ratio in the case of DNA is independent of the dye concentration —provided excess dye is present—and the binding is not influenced by the presence of low salt concentrations (< 0.3 M NaCl), the binding of azure A by DNPr can be increased considerably in the presence of salts, especially when higher concentrations of the dye are used. The possible implications of these results for the nature of the DNA-histone bond and for the use of cationic dyes for studies of nucleic acids at the tissue level are discussed.