Binding of the nonprotein chromophore of neocarzinostatin to deoxyribonucleic acid

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Abstract
The methanol-extracted, nonprotein chromophore of neocarzinostatin (NCS) [an antitumor agent], which has DNA-degrading activity comparable to that of the native antibiotic, had a strong affinity for DNA. Binding of chromophore was shown by DNA quenching of the 440-nm fluorescence and shifting of the emission peak to 420 nm; by DNA protection against spontaneous loss of activity in aqueous solution; and by DNA inhibition of the spontaneous generation of 490-nm fluorescence. Good quantitative correlation was found between these 3 methods in measuring chromophore binding. There was nearly a 1:1 correspondence between loss of chromophore activity and generation of 490-nm fluorescence; this suggests spontaneous degradation of active chromophore to a highly fluorescent product. Chromophore showed a preference for DNA high in adenine + thymine content in both fluorescence quenching and protection studies. NCS apoprotein, which is known to bind and protect active chromophore, quenched the 440-nm fluorescence, shifted the emission peak to 420 nm, and inhibited the generation of 490-nm fluorescence. Chromophore had a higher affinity for apoprotein than for DNA. Pretreatment of chromophore with 2-mercaptoethanol increased the 440-nm fluorescence 7-fold, and eliminated the tendency to generate 490-nm fluorescence. The 440-nm fluorescence of this inactive material was also quenched by DNA and shifted to 420 nm, indicating an affinity for DNA comparable to that of untreated chromophore. However, its affinity for apoprotein was much lower than that of untreated chromophore. Both 2-mercaptoethanol-treated and untreated chromophore unwound supercoiled pMB9 DNA, which suggests intercalation by both molecules. Since no physical evidence for interaction of native neocarzinostatin with DNA has been found, it is likely that dissociation of the chromophore from the protein and association with DNA are important steps in degradation of DNA by neocarzinostatin.

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