A Competition Assay for DNA Binding Using the Fluorescent Probe ANS

Fluoresence spectroscopy is a useful technique for investigating the interaction of DNA binding proteins with DNA. Generally, use is made of the intrinsic fluorescence of the protein arising from the aromatic amino acids, which is frequently perturbed in a DNA-protein complex (see Chapter 24 ). In some cases, however, changes in the intrinsic fluorescence emission of the protein arising from its interaction with nucleic acid may not be detectable. For example, if tryptophans and/or tyrosines are not located in the proximity of the DNA binding site, the emission spectrum may not be perturbed by the interaction. Furthermore, if the protein contains a large number of tryptophan and tyrosine residues, any effects may be masked because of the large number of aromatic residues whose emission spectrum is unperturbed by the binding of nucleic acid