J. Seetharamappa

Interaction of triprolidine hydrochloride with serum albumins: thermodynamic and binding characteristics, and influence of site probes.

The interaction between triprolidine hydrochloride (TRP) to serum albumins viz. bovine serum albumin (BSA) and human serum albumin (HSA) has been studied by spectroscopic methods. The experimental results revealed the static quenching mechanism in the interaction of TRP with protein. The number of binding sites close to unity for both TRP-BSA and TRP-HSA indicated the presence of single class of binding site for the drug in protein. The binding constant values of TRP-BSA and TRP-HSA were observed to be 4.75 ± 0.018 × 10(3) and 2.42 ± 0.024 × 10(4)M(-1) at 294 K, respectively. Thermodynamic parameters indicated that the hydrogen bond and van der Waals forces played the major role in the binding of TRP to proteins. The distance of separation between the serum albumin and TRP was obtained from the Försters theory of non-radioactive energy transfer. The metal ions viz., K(+), Ca(2+), Co(2+), Cu(2+), Ni(2+), Mn(2+) and Zn(2+) were found to influence the binding of the drug to protein. Displacement experiments indicated the binding of TRP to Sudlows site I on both BSA and HSA. The CD, 3D fluorescence spectra and FT-IR spectral results revealed the changes in the secondary structure of protein upon interaction with TRP.

Characterization of interaction and the effect of carbamazepine on the structure of human serum albumin.

The binding of carbamazepine (CBZ) to human serum albumin (HSA) was investigated under simulative physiological conditions. In this study, intrinsic fluorescence of tryptophan-214 in HSA was monitored upon the addition of CBZ. Binding constant of CBZ-HSA was calculated by the remarkable static quenching effect of CBZ and found to be (2.081+/-0.023)x10(4)M(-1). The fluorimetric results revealed that the hydrophobic interaction was a predominant intermolecular force for stabilizing the complex, which is also in agreement with the results obtained from voltammetric approach. Three site probes, warfarin, ibuprofen and digitoxin, were employed in fluorescence displacement experiments to locate the exact binding site for CBZ in HSA. The alteration in secondary structure of protein in the presence of CBZ was confirmed by the evidences from circular dichroism and FT-IR spectroscopy. Further, the distance r between donor (Trp-214) and acceptor (CBZ) was obtained according to fluorescence resonance energy transfer (FRET).

Interaction of antioxidant flavonoids with calf thymus DNA analyzed by spectroscopic and electrochemical methods

Mechanism of interaction of bioactive flavonoids, hesperitin (HES) and naringenin (NAR) with calf thymus deoxyribonucleic acid (DNA) was studied employing UV absorption, fluorescence, circular dichroism, melting temperature, fluorescence anisotropy and differential pulse voltammetric methods. The observed fluorescence quenching of DNA-ethidium bromide system by the flavonoid indicated the intercalative mode of binding between the flavonoid and DNA. Stern-Volmer plots have revealed the presence of static quenching mechanism. Binding and thermodynamic characteristics of interaction were evaluated. Melting temperature of DNA was found to be increased up to 5 °C in the presence of the flavonoid indicating the stabilization of DNA double helix upon binding. CD and fluorescence anisotropic results have revealed the conformational changes in DNA upon binding to the flavonoid. The observed positive shift in peak potential and decreased peak current of the flavonoid in the presence of DNA further supported the intercalative mode of binding.

Mechanism of interaction between human serum albumin and N-alkyl phenothiazines studied using spectroscopic methods

The binding characteristics of human serum albumin (HSA) with N-alkyl phenothiazines derivatives (NAP) viz., levomepromazine monomaleate (LMM) and propericiazine (PPC) have been studied by employing fluorescence, absorption, circular dichroism and FT-IR techniques. The Stern-Volmer quenching constant, K(SV) values were found to decrease with increase in temperature thereby indicating the presence of static quenching mechanism in the interactions of NAP with HSA. The number of binding sites, n and the binding constant, K were noticed to be, respectively, 1.11 and (5.188+/-0.034)x10(4) M(-1) for LMM and 1.06 and (4.436+/-0.066)x10(4) M(-1) for PPC at 298 K. The negative value of enthalpy change and positive value of entropy change in the present study indicated that the hydrophobic forces played a major role in the binding of NAP to HSA. The circular dichroism and FT-IR spectral data revealed the conformational changes in the structure of protein upon its interaction with NAP. The binding distances and the energy transfer efficiency between NAP and protein were determined based on Försters theory of energy transfer. The decreased binding constants of HAS-LMM and HAS-PPC systems in presence of common ions indicated the availability of higher concentration of free drug in plasma.

Spectral characterization of the binding and conformational changes of serum albumins upon interaction with an anticancer drug, anastrozole.

The present study employed different optical spectroscopic techniques viz., fluorescence, FTIR, circular dichroism (CD) and UV-vis absorption spectroscopy to investigate the mechanism of interaction of an anticancer drug, anastrozole (AZ) with transport proteins viz., bovine serum albumin (BSA) and human serum albumin (HSA). The drug, AZ quenched the intrinsic fluorescence of protein and the analysis of results revealed the presence of dynamic quenching mechanism. The binding characteristics of drug-protein were computed. The thermodynamic parameters, enthalpy change (ΔH°) and entropy change (ΔS°) were calculated to be +92.99 kJ/mol and +159.18 J/mol/K for AZ-BSA and, +99.43 kJ/mol and +159.19 J/mol/K for AZ-HSA, respectively. These results indicated that the hydrophobic forces stabilized the interaction between the drug and protein. CD, FTIR, absorption, synchronous and 3D fluorescence results indicated that the binding of AZ to protein induced structural perturbation in both serum albumins. The distance, r between the drug and protein was calculated based on the theory of Försters resonance energy transfer and found to be 5.9 and 6.24 nm, respectively for AZ-BSA and AZ-HSA.

RP‐HPLC Method for the Determination of Tenofovir in Pharmaceutical Formulations and Spiked Human Plasma

Abstract A simple reverse‐phase high‐performance liquid chromatographic method for the determination of tenofovir disoproxil fumarate (TDF) in pharmaceutical formulations and human plasma samples has been developed and validated. Piroxicam (PRX) was used as an internal standard. The assay of the drug was performed on a CLC C18 (5 μ, 25 cm×4.6 mm i.d.) with UV detection at 259 nm. The mobile phase consisted of acetonitrile–water mixture in the ratio of 75∶25, and a flow rate of 1 ml/min was maintained. The standard curve was linear over the range of 0.2–10 µg/ml (r 2=0.9966). Analytic parameters have been evaluated. Within‐day and between‐day precision as expressed by relative standard deviation was found to be less than 2%. The method has been applied successfully for the determination of TDF in spiked human plasma samples and pharmaceutical formulations.

Investigations to reveal the nature of interactions of human hemoglobin with curcumin using optical techniques

Curcumin (CUR) is an important bioactive compound present in the rhizome of Curcuma longa. Herein, we report the interaction of CUR with human hemoglobin (Hb) using various biophysical methods viz., fluorescence, UV absorption, resonance light scattering spectra (RLS), synchronous fluorescence, fluorescence anisotropy, circular dichroism (CD) and three-dimensional fluorescence. There was a considerable quenching of the intrinsic fluorescence of Hb upon binding to CUR through dynamic quenching mechanism. The distance (r) between the donor and acceptor was obtained from the Forsters theory of fluorescence resonance energy transfer (FRET) and found to be 1.55 nm. Alterations in the conformation of Hb due to its interaction with CUR were confirmed by UV absorption and CD spectroscopic methods. The α-helicity of Hb was found to decrease upon binding with CUR.

Electrochemical Oxidation of Bioactive Carbamazepine and Its Interaction with DNA

In the present work, the electrooxidation of carbamazepine (CBZ) was studied by employing cyclic, linear, and differential pulse voltammetric (DPV) techniques. The dependence of peak current and peak potential on scan rate, pH, temperature, concentration, surfactant, and different electrolytes was examined. The interaction between CBZ and calf thymus DNA (ctDNA) was investigated. From electrochemical data, the binding constant of CBZ-ctDNA was calculated and the intercalative mode of binding was proven. A DPV method with good precision and accuracy was developed for the determination of CBZ in bulk samples and tablets.

Voltammetric sensor for buzepide methiodide determination based on TiO2 nanoparticle-modified carbon paste electrode

In this work, we have prepared nano-material modified carbon paste electrode (CPE) for the sensing of an antidepressant, buzepide methiodide (BZP) by incorporating TiO2 nanoparticles in carbon paste matrix. Electrochemical studies indicated that the TiO2 nanoparticles efficiently increased the electron transfer kinetics between drug and the electrode. Compared with the nonmodified CPE, the TiO2-modified CPE greatly enhances the oxidation signal of BZP with negative shift in peak potential. Based on this, we have proposed a sensitive, rapid and convenient electrochemical method for the determination of BZP. Under the optimized conditions, the oxidation peak current of BZP is found to be proportional to its concentration in the range of 5 x 10(-8) to 5 x 10(-5)M with a detection limit of 8.2 x 10(-9)M. Finally, this sensing method was successfully applied for the determination of BZP in human blood serum and urine samples with good recoveries.

Interaction of an antidepressant buzepide methiodide with DNA immobilized on the glassy carbon electrode

In the present study, a DNA-biosensor was prepared using immobilization technique to investigate the interaction between an antidepressant, buzepide methiodide (BZP) and calf thymus DNA. BZP showed a quasireversible peak in Britton-Robinson (BR) buffer of pH 5 at bare glassy carbon electrode (GCE). At DNA modified GCE, the peak potential of BZP was observed to be shifted towards positive potential revealing intercalative mode of binding. The binding constant and stoichiometry between DNA and BZP are calculated to be 1.908×10(5)M(-1) and 0.982, respectively. The spectroscopic techniques viz., spectrofluorescence and UV-vis absorption have also been employed to understand the interaction between BZP and DNA. The results serve as a reference for the interaction of BZP with DNA base pairs in the natural environment of living cells.