Junhui Pan

Spectroscopic Studies of DNA Interactions with Food Colorant Indigo Carmine with the Use of Ethidium Bromide as a Fluorescence Probe

The interaction of indigo carmine (IC) with calf thymus DNA in physiological buffer (pH 7.4), using ethidium bromide (EB) dye as a fluorescence probe, was investigated by ultraviolet-visible absorption, fluorescence, and circular dichroism (CD) spectroscopy, coupled with viscosity measurements and DNA-melting studies. Hypochromicity of the absorption spectra of IC and enhancement in fluorescence polarization of IC were observed with the addition of DNA. Moreover, the binding of IC to DNA was able to decrease iodide and single-stranded DNA (ssDNA) quenching effects, increase the melting temperature and relative viscosity of DNA, and induce the changes in CD spectra of DNA. All of the evidence indicated that IC interacted with DNA in the mode of intercalative binding. Furthermore, the three-way synchronous fluorescence spectra data obtained from the interaction between IC and DNA-EB were resolved by parallel factor analysis (PARAFAC), and the results provided simultaneously the concentration information and the pure spectra for the three reaction components (IC, EB, and DNA-EB) of the system at equilibrium. This PARAFAC demonstrated that the intercalation of IC molecules into DNA proceeded by substituting for EB in the DNA-EB complex. The calculated thermodynamic parameters, ΔH° and ΔS°, suggested that both hydrophobic interactions and hydrogen bonds played a predominant role in the binding of IC to DNA.

Groove binding interaction between daphnetin and calf thymus DNA

The binding characteristics of daphnetin with calf thymus DNA (ctDNA) were investigated by multispectroscopic and chemometric approaches coupled with DNA viscosity measurements, melting studies and molecular docking technique. The expanded UV-vis spectral data matrix was processed by multivariate curve resolution-alternating least-squares method to obtain the concentration profiles of the components (daphnetin, ctDNA and daphnetin-ctDNA complex) to quantitatively monitor the daphnetin-ctDNA interaction. The groove mode of daphnetin binding to ctDNA was concluded by little change in melting temperature, viscosity of ctDNA and iodide quenching effect as well as increase in single-stranded DNA quenching effect. Moreover, the quantitative data for the competitive binding between daphnetin and Hoechst 33258 for ctDNA obtained by resolving the three-way synchronous fluorescence spectra data using parallel factor analysis modeling further supported the groove binding. The molecular docking visualized the results of the Fourier transform infrared analysis that the adenine and thymine bases in the minor groove of ctDNA were the main binding sites for daphnetin, and the circular dichroism spectra showed that the groove binding of daphnetin to ctDNA led to the conformational change in ctDNA from B-form to A-form. This study revealed the interaction mechanism of daphnetin with ctDNA.

Inhibition of chrysin on xanthine oxidase activity and its inhibition mechanism.

Chrysin, a bioactive flavonoid, was investigated for its potential to inhibit the activity of xanthine oxidase (XO), a key enzyme catalyzing xanthine to uric acid and finally causing gout. The kinetic analysis showed that chrysin possessed a strong inhibition on XO ability in a reversible competitive manner with IC50 value of (1.26±0.04)×10(-6)molL(-1). The results of fluorescence titrations indicated that chrysin bound to XO with high affinity, and the interaction was predominately driven by hydrogen bonds and van der Waals forces. Analysis of circular dichroism demonstrated that chrysin induced the conformational change of XO with increases in α-helix and β-sheet and reductions in β-turn and random coil structures. Molecular simulation revealed that chrysin interacted with the amino acid residues Leu648, Phe649, Glu802, Leu873, Ser876, Glu879, Arg880, Phe1009, Thr1010, Val1011 and Phe1013 located within the active cavity of XO. The mechanism of chrysin on XO activity may be the insertion of chrysin into the active site occupying the catalytic center of XO to avoid the entrance of xanthine and causing conformational changes in XO. Furthermore, the interaction assays indicated that chrysin and its structural analog apigenin exhibited an additive effect on inhibition of XO.

Intercalation binding of food antioxidant butylated hydroxyanisole to calf thymus DNA

The binding properties of food antioxidant butylated hydroxyanisole (BHA) associated with calf thymus DNA (ctDNA) in physiological buffer (pH 7.4) were investigated. Experimental results based on fluorescence, UV-vis absorption, circular dichroism (CD), viscosity measurements and autodocking techniques confirmed the intercalation binding between BHA and ctDNA. The changes in Fourier transform infrared spectra of ctDNA induced by BHA suggested that BHA was more prone to bind to G-C rich region of ctDNA, which was further ascertained with the molecular docking studies. Analysis of the CD spectra indicated that this binding interaction led to a transformation from B-like DNA structure toward A-like conformation. The complexation of BHA with ctDNA was driven mainly by hydrogen bonds and hydrophobic forces. The binding constants of the BHA-ctDNA complex were calculated to be 2.03 × 10(4), 1.92 × 10(4) and 1.59 × 10(4)L mol(-1) at 298, 304 and 310 K, respectively. Gel electrophoresis results suggested that intercalated BHA molecules did not significantly affect plasmid DNA. Moreover, the concentration profiles and the spectra for the three reaction components (BHA, ctDNA, and BHA-ctDNA complex) of the system by resolving the augmented UV-vis spectral data matrix with the use of multivariate curve resolution-alternating least squares approach provided quantitative data to estimate the progress of BHA-ctDNA interaction. This study is expected to provide new insights into the mechanism of interaction between BHA and ctDNA.

Binding properties of butylated hydroxytoluene with calf thymus DNA in vitro.

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)×10(3), (2.86±0.02)×10(3) and (1.80±0.04)×10(3) 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.

Characterization of the interaction between resmethrin and calf thymus DNA in vitro

Resmethrin (RES) is a synthetic pyrethroid insecticide widely used to control pests in agriculture, but it may cause potential hazards to human health. The characteristics of the binding of RES with calf thymus DNA (ctDNA) were investigated in a physiological buffer (pH 7.4) by multiple spectroscopic methods combined with ctDNA melting and viscosity measurements, multivariate curve resolution-alternating least-squares (MCR-ALS) chemometrics and the molecular docking technique. The concentration profiles and the pure spectra for the reactive species (RES, ctDNA and RES–ctDNA complex), obtained through decomposing the augmented UV-vis spectral data matrix by the MCR-ALS approach, indicated that RES could bind to ctDNA and the reaction process could be quantitatively monitored. The RES molecules bound to ctDNA by groove binding, as evidenced by the negligible changes in the ctDNA melting temperature, viscosity and iodide quenching effect and the increase in the single-stranded DNA quenching effect. Fluorescence titration data indicated that the complexation of RES with ctDNA was mainly driven by hydrogen bonds and van der Waals forces, and had strong affinity. The changes in the Fourier transform infrared spectra of ctDNA suggested that RES molecules preferentially bound to the G–C region of ctDNA, which was consistent with the prediction of the molecular docking. The circular dichroism spectral analysis indicated that RES induced a decrease in the right-handed helicity of ctDNA. The DNA cleavage assay showed that RES did not cleave the pUC18 plasmid DNA. This study offers a comprehensive picture of RES–ctDNA interaction, which may provide insights into the toxicological effect of the insecticide.

Deciphering the inhibitory mechanism of genistein on xanthine oxidase in vitro.

Genistein (Gen), widely distributed in soybean, is proved to be important in homeostasis in the human body. Herein, the inhibitory mechanism of Gen against xanthine oxidase (XO) was studied through multispectroscopic methods and molecular simulation. The inhibition kinetics showed that Gen competitively inhibited XO with an inhibition constant of (1.39 ± 0.11) μM by competing with xanthine for binding to the active site of XO. Fluorescence titration study suggested that the fluorescence quenching mechanism of XO was static, resulting from the formation of a Gen-XO complex at one fold site. The calculated thermodynamic parameters revealed that the interaction process was driven mainly by hydrophobic interactions and hydrogen bonds with affinity of (5.24 ± 0.02) × 10(4) Lmol(-1). Conformational analyses demonstrated that the microenvironment and the secondary structure of XO were changed upon binding of Gen. The molecular docking displayed that Gen bound to the active cavity of XO by interacting with the surrounding amino acid residues (Leu648, Phe649, Glu802, Ser876, Glu879, Arg880, Phe914, Phe1009, Thr1010 and Phe1013). Thus, the inhibition may be attributed to the insertion of Gen into the active site of XO occupying the catalytic center of the enzyme to avoid entry of the substrate and inducing conformational changes of XO (more compact), which was further unfavorable for forming the active cavity and further reduced the landing and oxidation of substrate. This study may offer novel insights into the inhibition mechanism of Gen on XO.

Galangin competitively inhibits xanthine oxidase by a ping-pong mechanism

Galangin is a natural flavonol isolated from plants with potent biological activities. Galangin was found to significantly inhibit xanthine oxidase (XOD) activity in a competitive manner with the generation of superoxide radical (O2-) in the enzyme catalysis, but galangin showed insignificant scavenging activity on 1,1-diphenyl-2-picryhydrazyl (DPPH) and O2- radicals. These results demonstrated that inhibition of O2- radical generation by galangin may be due to the competitive inhibition of uric acid formation by a ping-pong mechanism. XOD had one high affinity binding site for galangin with a binding constant of 3.60×104Lmol-1 at 298K. Hydrogen bond and hydrophobic interaction dominated the binding process on account of the negative enthalpy and positive entropy changes. The binding of galangin to XOD induced an increase in α-helix and random coil contents and a decrease in β-sheet and β-turn contents of XOD. Further molecular docking study validated that galangin can competitively bind to the site in the molybdenum atomic (Mo) domain, occupying the catalytic center to avoid the entrance of the substrate xanthine, resulting in the inhibition of XOD activity. These findings have provided new insights into the two-substrate kinetics of galangin on XOD and useful information for functional research of galangin in the treatment of gout and oxidative damage.

Characterization of the groove binding between di-(2-ethylhexyl) phthalate and calf thymus DNA

In this study, the interaction between di-(2-ethylhexyl) phthalate (DEHP) and calf thymus DNA (ctDNA) was investigated by a combination of multispectroscopic methods, chemometrics algorithm, cyclic voltammetry and molecular simulation. The concentration profiles of the components obtained from resolving the UV-vis absorption data by multivariate curve resolution-alternating least-squares (MCR-ALS) provided a basic evidence for the formation of DEHP-ctDNA complex. Furthermore, the groove binding of DEHP to ctDNA was evidenced by the results from iodide quenching effect, single-stranded DNA quenching effect, melting studies, viscosity measurements and cyclic voltammetry. The binding constant of the complex was in the order of magnitudes of 104Lmol-1, and hydrophobic forces were inferred to drive the binding process. Analysis of Fourier transform infrared spectra suggested that DEHP preferentially bound to A-T rich region of ctDNA in the minor groove, and these results further confirmed by molecular docking. The circular dichroism spectra indicated that DEHP induced a decrease in base stacking degree and an increase in right-handed helicity of ctDNA, but did not cause a significant damage in DNA. This study may improve the understanding of interaction between DEHP and ctDNA and help evaluate the toxicological effect of DEHP.

Intercalation of 2-butyl-4-methylphenol to G-C rich region of DNA and the role of hydroxypropyl-β-cyclodextrin.

The characteristics of binding of 2-tert-butyl-4-methylphenol (TBMP), a synthetic phenolic antioxidant with hydroxypropyl-β-cyclodextrin (Hp-βCD) and calf thymus DNA (ctDNA) were investigated by multi-spectroscopic techniques and molecular simulation. The results indicated that TBMP preferred to form a 1:1 inclusion complex with Hp-βCD, with an inclusion constant of determined to be 7.15 × 10(3) L mol(-1). The intercalative mode of TBMP to ctDNA was supported by ctDNA melting temperature and relative viscosity studies, salt quenching effect, competitive binding with methylene blue and molecular modeling. The changes in Fourier transformed infrared (FT-IR) and circular dichroism (CD) spectra suggested that TBMP mainly bound to the G-C rich region with inducing a significant perturbation in B-like DNA structure. It was also found that Hp-βCD decreased the binding ability of TBMP with ctDNA, but did not affect the interactive mode between TBMP and ctDNA, and the formed inclusion complex of TBMP-Hp-βCD decomposed in the presence of ctDNA. This study may provide insights into the mechanism of binding of TBMP with ctDNA and the role of Hp-βCD in the TBMP-ctDNA interaction.