| Summary: | 碩士 === 國立彰化師範大學 === 化學系 === 93 === Abstract
Neocarzinostatin (NCS) is a potent antitumor antibiotic complex comprised of a DNA cleaving agent, an enediyne chromophore, and an all β-sheet apo-neocarzinostatin. The apo-neocarzinostatin contributes to the stabilization and regulation of the labile chromophore when the DNA cleavage role of NCS is required. The factors pertaining to the delicate balance between the drug stabilization, which involves the drug-protein binding, and the drug activity, which involves the drug-DNA binding, are not fully understood.
Earlier it was reported that sodium chloride inhibited the NCS-mediated DNA strand scission in the presence and absence of a thiol. One of the possible causes could be resulted from salt-protein interactions that affects the protein function pertaining to stabilization and regulation of NCS chromophore. The other possible causes include the salt effect on salt-DNA interactions, which could interfere the binding of NCS chromophore to the target DNA. The present study is intended to understand the mechanism of salt inhibition of NCS-mediated DNA cleavage.
Series of in vitro DNA cleavage experiments are done under different salt concentrations in the presence of the thiols, glutathione or 2-mercaptoethanol. The results show that the degree of inhibition of NCS-mediated DNA cleavage by sodium chloride with either holoNCS or free NCS chromophore is similar to each other. These results indicate that salt-protein interaction may not be the major inhibitory factor. The measurements of Tm values of apoNCS in the presence of sodium chloride, monitored by Far-UV CD analyses at 224 nm, reveal that sodium chloride does not alter the thermal stability of apo-neocarzinostatin. To examine whether the inhibitory effect of sodium chloride is based on the possible influence of sodium chloride on the protein, fluorescence analyses of the chromophore release kinetics were performed in the presence of the salt. The kinetic results show modest salt effect, indicating that the salt-protein interaction is not a major cause for the inhibition on the drug activity.
NCS chromophore-DNA binding constant in the presence of salts were estimated by measuring the rate of chromophore degradation in the presence and absence of DNA. The rate of degradation was determined by the appearance of 490 nm fluorescence from the bound and dissociated chromophore in the presence of different concentration of salts. The changes of NCS chromophore-DNA binding free energy change (ΔG°) with salt shows high order of corroboration with the extent of inhibition by salt in the drug-induced DNA cleavage. Altogether, the results of the present study suggest that the major inhibitory effect of salts on NCS-mediated DNA cleavage is not from salt-protein interactions or salt-chromophore interaction but from the prevention of binding of NCS chromophore to DNA.
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