Ixing was given and absorbance was subsequently recorded at 260 nm [39]. In pH melting study the absorbance reached a plateau by ,18?0 ml of 1 M NaOH. The pH that corresponded to the midpoint 
between the initial and final absorbance values was taken as the melting pH. Accordingly, the melting point was found to be 11.9560.01. The pH melting profile was obtained for a) DNA alone b) DNA with drugs at varying P/D ratios and the percentage of hyperchromicity 18325633 was computed (at each point of pH varying from 1.9 
to 19.9) using the formula 100 (A2602Ao260)/ (A260max2Ao260), where A260 is absorbance at 260 nm at any particular pH, Ao260 is the initial absorbance at 260 nm 12926553 and A260max is the maximum absorbance attained after reaching plateau.where the CDM, CD, and CM are the analytical concentrations of DNA-methylxanthines complex, DNA and methylxanthines (theophylline or theobromine or caffeine) respectively. The Beer Lambert law for the absorption of light is assumed to be followed by the DNA drug binding. CD CD0 {CDM ??CDM and(A{A0 ) eDM :L??CD0A0 eD : L??FTIR spectroscopyFTIR spectroscopy was IQ1 cost employed to study the mode of interaction of theophylline, theobromine and caffeine both in the presence or absence of Mg2+ (1?0 mM) with Herring sperm DNA (not highly polymerized). DNA-drug and Mg2+-DNA-drugs complexes were prepared and the spectra were obtained with repeated scanning between 1400?00 cm21 according to our published protocol [40].where CD0 is the concentration of pure DNA, A0 and A are the absorbance (at 260 nm) of pure DNA and in the presence of methylxanthines (theophylline or theobromine or caffeine) respectively. L is the path length of the cuvette i.e. 1 cm. By incorporating the values of CD and CDM from the above equations into equation (3), the following equation can be derived: A0 eD eD 1 | z (A{A0 ) eDM eDM :K M Therefore the double reciprocal plot of 1/(A2A0) versus 1/[CM] is linear and the binding 56-59-7 web constant (K) can be estimated by calculating the ratio of the intercept to the slope, K Intercept Slope ??Results and Discussion Interaction of methylxanthines with native form of DNA: UV absorptionWe examined the changes induced in the UV spectra of calf thymus DNA owing to interaction of xanthine derivatives at different P/D ratios (0.8, 1.0, 3.0 and 6.0). The ultraviolet absorbance for free methylxanthines, free DNA and DNAmethylxanthines complexes were obtained. Based on these spectra, percentages of hyperchromicity (Fig. 2A) and the binding constants of methylxanthines with DNA (Figs. 2B ) were calculated as described. Figures 2A confirm the binding of methylxanthines with DNA, and the binding affinity increased with respect to increasing drug concentration, exhibiting a dose dependent behavioral pattern for DNA binding. The percentage of hyperchromicity indicates a similar fashion or mode ofScheming of hyperchromicityThe percentage of hyperchromicity was computed for the binding interaction of xanthine derivatives with DNA at different P/D’s using the formula: 100(A2602Ao260)/(A260max2Ao260), whereMethylxanthines Binding with DNAmethylxanthines binding with DNA bases. Though the calculated hyperchromicity denotes a similar mode of binding (Fig. 2A), these methylxanthines exhibit a differential binding efficacy with DNA, where caffeine and theophylline show up little higher binding efficacy with DNA than theobromine as predicted by binding constant analysis (Figs. 2B ). Thus the order of binding affinity is visualized as.Ixing was given and absorbance was subsequently recorded at 260 nm [39]. In pH melting study the absorbance reached a plateau by ,18?0 ml of 1 M NaOH. The pH that corresponded to the midpoint between the initial and final absorbance values was taken as the melting pH. Accordingly, the melting point was found to be 11.9560.01. The pH melting profile was obtained for a) DNA alone b) DNA with drugs at varying P/D ratios and the percentage of hyperchromicity 18325633 was computed (at each point of pH varying from 1.9 to 19.9) using the formula 100 (A2602Ao260)/ (A260max2Ao260), where A260 is absorbance at 260 nm at any particular pH, Ao260 is the initial absorbance at 260 nm 12926553 and A260max is the maximum absorbance attained after reaching plateau.where the CDM, CD, and CM are the analytical concentrations of DNA-methylxanthines complex, DNA and methylxanthines (theophylline or theobromine or caffeine) respectively. The Beer Lambert law for the absorption of light is assumed to be followed by the DNA drug binding. CD CD0 {CDM ??CDM and(A{A0 ) eDM :L??CD0A0 eD : L??FTIR spectroscopyFTIR spectroscopy was employed to study the mode of interaction of theophylline, theobromine and caffeine both in the presence or absence of Mg2+ (1?0 mM) with Herring sperm DNA (not highly polymerized). DNA-drug and Mg2+-DNA-drugs complexes were prepared and the spectra were obtained with repeated scanning between 1400?00 cm21 according to our published protocol [40].where CD0 is the concentration of pure DNA, A0 and A are the absorbance (at 260 nm) of pure DNA and in the presence of methylxanthines (theophylline or theobromine or caffeine) respectively. L is the path length of the cuvette i.e. 1 cm. By incorporating the values of CD and CDM from the above equations into equation (3), the following equation can be derived: A0 eD eD 1 | z (A{A0 ) eDM eDM :K M Therefore the double reciprocal plot of 1/(A2A0) versus 1/[CM] is linear and the binding constant (K) can be estimated by calculating the ratio of the intercept to the slope, K Intercept Slope ??Results and Discussion Interaction of methylxanthines with native form of DNA: UV absorptionWe examined the changes induced in the UV spectra of calf thymus DNA owing to interaction of xanthine derivatives at different P/D ratios (0.8, 1.0, 3.0 and 6.0). The ultraviolet absorbance for free methylxanthines, free DNA and DNAmethylxanthines complexes were obtained. Based on these spectra, percentages of hyperchromicity (Fig. 2A) and the binding constants of methylxanthines with DNA (Figs. 2B ) were calculated as described. Figures 2A confirm the binding of methylxanthines with DNA, and the binding affinity increased with respect to increasing drug concentration, exhibiting a dose dependent behavioral pattern for DNA binding. The percentage of hyperchromicity indicates a similar fashion or mode ofScheming of hyperchromicityThe percentage of hyperchromicity was computed for the binding interaction of xanthine derivatives with DNA at different P/D’s using the formula: 100(A2602Ao260)/(A260max2Ao260), whereMethylxanthines Binding with DNAmethylxanthines binding with DNA bases. Though the calculated hyperchromicity denotes a similar mode of binding (Fig. 2A), these methylxanthines exhibit a differential binding efficacy with DNA, where caffeine and theophylline show up little higher binding efficacy with DNA than theobromine as predicted by binding constant analysis (Figs. 2B ). Thus the order of binding affinity is visualized as.
