Phase partition studies of actinomycin-nucleotide complexes.

A method is reported for measuring the stoichiometry of complex formation between actinomycin and a series of deoxynucleotides. The amount of bound actinomycin is measured by distribution of the drug between two liquid phases, a buffer phase containing deoxynucleotide and an organic phase in which the nucleotide is insoluble. Using simple statistical mechanical analysis, the equilibrium equations for several models of actinomycin-deoxynucleotide complexes have been derived: actinomycin with one binding site, with two equivalent independent binding sites, and with two sites which must be occupied together. The binding of actinomycin C3 with dpG, dpApG, dpA, and dpGpC has been examined compared with these models. It is found that binding to dpG and dpApG involves two independent binding sites of nearly equal affinity for nucleotides, whereas binding of dpGpC to the two binding sites on actinomycin is a cooperative process. Binding of dpA tp actinomycin is partially cooperative and weaker than binding of dpG. The dimerization constant of actinomycin was also determined by the phase separation technique, and found in agreement with other values, including the results of kinetic measurements reported here.