Phase partition studies of actinomycin-nucleotide complexes

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Abstract
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 2 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 were derived: actinomycin with 1 binding site, with 2 equivalent independent binding sites and with 2 sites which must be occupied together. The binding of actinomycin C3 with dpG, dpApG, dpA and dpGpC was examined and compared with these models. Binding to dpG and dpApG involves 2 independent binding sites of nearly equal affinity for nucleotides, whereas binding of dpGpC to the 2 binding sites on actinomycin is a cooperative process. Binding of dpA to 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.
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