Binding properties of butylated hydroxytoluene with calf thymus DNA in vitro

Abstract

The binding properties of butylated hydroxytoluene (BHT) with calf thymus DNA (ctDNA) in simulated physiological buffer (pH 7.4) were investigated using ethidium bromide (EB) dye as a fluorescence probe by various spectroscopic techniques including UV–vis absorption, fluorescence, circular dichroism (CD), and Fourier transform infrared (FT-IR) spectroscopy along with ctDNA melting studies and viscosity measurements. It was found that the binding of BHT to ctDNA could decrease the absorption intensity of ctDNA, significantly increase melting temperature and relative viscosity of ctDNA, and induce the changes in CD spectra. Moreover, the competitive binding studies showed that BHT was able to displace EB from the bound ctDNA–EB complex. All the experimental results indicated that the binding mode between BHT and ctDNA was an intercalation. The association constants between BHT and ctDNA were evaluated to be (4.78±0.04)×103, (2.86±0.02)×103 and (1.80±0.04)×103 L mol−1 at 298, 304, 310K, respectively. Further, the FT-IR analysis revealed that BHT was more prone to interact with adenine and thymine base pairs, and no significant conformational transition of ctDNA occurred. Thermodynamic analysis of the binding data showed that the binding process was primarily driven by hydrogen bonds and van der Waals forces, as the values of the enthalpy change and the entropy change were calculated to be −62.47±0.07kJ mol−1 and −139.22±0.22J mol−1 K−1, respectively.