Molecular Pharmacology, Vol 3, 327-332, Copyright © 1967 by the American Society for Pharmacology and Experimental Therapeutics

Methods for Assaying Complexes between Acridine Orange and Deoxyribonucleic Acid

¹ Department of Genetics, University of Pavia, Pavia, Italy

In acridine orange—DNA complexes (AO-DNA), the weak (external) complex can be distinguished from the strong (intercalated) one by hot dialysis and by fluorescence spectra. It has been found that the weak complex dissociates at low temperatures (<T_m) whereas the strong complex dissociates only during DNA denaturation. Hot dialysis was carried out at temperatures T such that 50° < T < T^* (T^* = temperature at which the melting starts), thus retaining only the intercalated complex. Subsequent T_m determinations of the strong complex yielded a value of (bound dye/nucleotide) saturation ratio (r_sat) of about 0.11.

Fluorescence spectra, excited by UV, of AO-DNA (native) and AO-DNA (denatured) complexes indicate that the peak at 515 mµ is due to the strong complex and the peak at 620 mµ to the weak complex. The intensity of the 515 mµ peak reaches a maximum at a value of r_sat of about 0.11.

Note:
ACKNOWLEDGMENTS Thanks are expressed to Professor E. Testa (Lepetit Corp.) for purifying the acridine orange, and to Professors P. Caldirola and R. Fieschi for allowing us to use some of their instruments. Miss A. Uggé and Miss M. Berti gave very valuable technical assistance. Work supported by Assegnazione No. 04/130/ 5/1298 of the Consiglio Nazionale delle Ricerche.