The related antibiotics rubiflavin and hedamycin interact similarly with deoxyribonucleic, acid in vitro. Formation of strong deoxyribonucleic acid-antibiotic complexes brings about a number of changes in the physical and chemical properties of the deoxyribonucleic acid. As a result of complex formation the visible absorption spectra of the antibiotics are red shifted and depressed, with development of shoulders suggesting that the antibiotic chromophores are in a hydrophobic environment. The temperature of the deoxyribonucleic acid melting transition is increased in the presence of the antibiotics, and enzymatic digestion of native deoxyribonucleic acid by snake venom phosphodiesterase is strongly inhibited. Exhaustive dialysis of Escherichia coli deoxyribonucleic acid-rubiflavin complexes showed that at low ionic strength the ratio of tightly bound rubiflavin molecules per deoxyribonucleic acid nucleotide is 0.53, while for hedamycin the ratio is 0.20. Denaturation of deoxyribonucleic acid did not greatly affect the tight binding of rubiflavin (ratio 0.57), but high ionic strength reduced it (ratio 0.23). Raising the percentage guanosine plus cytosine by using Micrococcus lysodeikticus deoxyribonucleic acid had little effect on the amount of tight binding (ratio 0.57), while for poly dAT, binding was decreased significantly (ratio 0.35). Complexes of DNA with rubiflavin and hedamycin band in CsCl gradients at positions of considerably lower density than the uncomplexed DNA. Mixtures of the complexes with the uncomplexed DNA in CsCl solution form separate bands upon centrifugation, indicating that equilibration of antibiotic molecules among individual DNA molecules is not extensive. The binding is probably nOncovalent, since rubiflavin can be extracted with benzene from an aqueous solution of the complex at high ionic strength. At least three kinds of binding to polynucleotides were recognized: (a) a strong ionic type prominent in complexes of the antibiotics with the homopolynucleotides poly U, poly C, and poly A; this binding was reversible to extensive dialysis at high ionic strength; (b) an effectively irreversible type found in all DNA-antibiotic complexes studied; this type was partially reversed by dialysis at high ionic strength, and was characterized by the appearance of shoulders in the visible spectrum of the antibiotics; (c) an interaction of antibiotic molecules with one another to cause their aggregation on the surface of DNA. Hedamycin, which is more potent than rubiflavin as a bactericidal agent, causes a much greater change in the properties of DNA per molecule bound than does rubiflavin, although the maximum number of hedamycin molecules that can bind per DNA nucleotide is only about half that of rubiflavin. The strong affinity of these antibiotics for DNA suggests an antibacterial mechanism in which rubiflavin and hedamycin become intracellularly bound to DNA and prevent DNA synthesis by inhibiting strand separation. © 1969, American Chemical Society. All rights reserved.
