Studies of protein-DNA interactions by capillary electrophoresis/laser-induced fluorescence polarization

Protein-DNA interactions were studied on the basis of capillary electrophor etic separation of bound from free fluorescent probe followed by on-line de tection with laser-induced fluorescence polarization. Changes in electropho retic mobility and fluorescence anisotropy upon complex formation were moni tored for the determination of binding affinity and stoichiometry. The meth od was applied to study the interactions of single-stranded DNA binding pro tein (SSB) with synthetic oligonucleotides and single-stranded DNA. Increas es in fluorescence anisotropy and decreases in electrophoretic mobility upo n their binding to SSB were observed for the fluorescently labeled 11-mer a nd 37-mer oligonucleotide probes, Fluorescence anisotropy and electrophoret ic mobility were used to determine the binding constants of the SSB with th e 11-mer (5 x 10(6) M-1) and the 37-mer (23 x 10(6) M-1). Alternatively, a fluorescently labeled SSB was used as a probe, and the formation of multipl e protein-DNA complexes that differ in stoichiometry was observed. The resu lts demonstrate the applicability of the method to study complex interactio ns between protein and DNA.