Interactions of the DNA intercalator acridine orange, with itself, with caffeine, and with double stranded DNA

Caffeine (CAF) inhibits the intercalation of acridine orange (AO) into cellular DNA. Optical absorption and fluorescence spectroscopy were employed to determine the molecular interactions of AO with itself, with CAF, and with double stranded herring sperm DNA (dsDNA). AO dimerization was observed at concentrations > 2 mumol. The sharp increase in fluorescence (lambda(cm) = 530 nm) at 5 mumol of AO was attributed to AO multimer formation. From 0.5 to 5.0 mumol, the AO self-association binding constant (K-assoc) was determined to be 3 8 620 mol(-1), however, the presence of 150 mmol NaCl increased K-assoc to 118 000 mol(-1) attributed to the charge neutralization. The K-assoc for AO with CAF was confirmed to be 256 mol(-1). K-assoc for the binding of AO with 20 mumol DNA ranged from, 32 000 mol(-1) at 2 mumol AO, to approximately 3700 mol(-1) at 10 mumol AO, in the absence of NaCl. This AO concentration dependency of K-assoc value with DNA was attributed to AO intercalation into dsDNA at high dsDNA/ AO ratios, and electrostatic binding of AO to dsDNA at low AO ratios. The findings provide information used to explain fluorescence intensity values at lambda(cm) at 530 nm from studies that combine AO, caffeine, and dsDNA. (C) 2002 Elsevier Science B.V. All rights reserved.