| Abstract: | The nonprotein chromophore of neocarzinostatin was found to share many of the characteristics of classical intercalators in its interaction with DNA. Viscosity studies with PM2 DNA indicated that the DNA helix unwinding induced by the chromophore was 0.82 times that of ethidium or 21 degrees. Electric dichroism of the chromophore--DNA complex showed that each bound chromophore molecule lengthened DNA by 3.3 A and that absorbance transitions of the chromophore at 315--385 nm were oriented approximately parallel to DNA bases, as expected for an intercalated aromatic ring. Binding to DNA induced strong hypochromicity and a pronounced red shift in the absorbance spectrum of the chromophore. Spectrophotometric titrations suggested at least two types of chromophore binding sites on DNA; one type of site was saturated at rb = 0.125 chromophore molecule/nucleotide, but binding to additional sites continued to at least rb = 0.3. These physical--chemical studies were performed at pH 4--5 in order to keep the chromophore stable, but chromophore bound to an excess of DNA at pH 7 showed a stable absorbance spectrum identical with that seen at pH 4--5, suggesting that a similar type of binding occurs at neutral pH. Chromophore which had spontaneously degraded in pH 8 buffer did not bind to DNA at all, as judged by absorbance spectroscopy. The degree of protection afforded by DNA against spontaneous chromophore degradation implied a dissociation constant of approximately 5 microM for the DNA--chromophore complex at neutral pH and physiological ionic strength. Supercoiled DNA was nearly twice as effective as relaxed DNA in protecting chromophore from degradation, providing additional evidence for intercalation at neutral pH. Comparison of absorbance, fluorescence, and dichroism spectra suggests that the naphthalene ring system is the intercalating moiety. |
