Analysis of Binding of Daunorubicin and Doxorubicin to DNA Using Computerized Curve-Fitting Procedures

Abstract

The direct and difference spectroscopic methods of determining the binding constant of daunorubicin to DNA were compared. In addition, several other experimental parameters were varied, including the cell path length (1 or 10cm), solution concentration, and analytical wavelength. The criterion used for judging the conditions and experimental methods was the attainment of a well-defined binding isotherm. Computerized least-squares curve fitting was used to determine the DNA binding constants from the binding isotherms and to compare the fit of a one-site and two-site model to the data. These studies showed that the best technique for determining the daunorubicin-DNA binding constants involved difference spectroscopic analysis of the absorbance change at 480nm in long path length cells. In addition, overnight equilibration of daunorubicin-DNA complexes produced spectral changes, which indicated that methods involving prolonged equilibration would be inappropriate for daunorubicin-DNA binding studies. Computerized curve fitting of the data obtained from daunorubicin-DNA and doxorubicin-DNA solutions gave DNA binding constants of 1.27×106M−1 for daunorubicin and 2.04×106M−1 for doxorubicin and showed that a one-site model fit the data better than a two-site model. These binding constants are consistent with other physical and biological data and provide a very preliminary indication that DNA affinity and biological activity (e.g., antitumor activity) may be related in the anthracyclines. In addition, the value of the number of binding sites obtained from the computerized curve-fitting program was 8/40 or 0.2, which is in agreement with the neighbor exclusion model. The methods and criterion used in this study describe a general approach for determining accurately the binding of drugs to DNA.