Debi Ranjan Tripathy

The influence of common metal ions on the interactions of the isoflavone genistein with bovine serum albumin.

The interaction of genistein with bovine serum albumin (BSA) has been characterized via UV-vis, fluorescence spectroscopy and Circular Dichroism (CD) measurements under physiological conditions. In this study, we have investigated the effect of some common metal ions on the binding of genistein with BSA using fluorescence studies. The fluorescence data reveal that the binding affinity of genistein to BSA increases in presence of certain metal ions. The possibility of non-radiative energy transition from the donor tryptophan to the acceptor genistein has been observed in absence and presence of metal ions. The observed similarities in the values of efficiency of energy transfer (E) and the separation between the donor and acceptor (r) in both the cases may be correlated with the complexation between the genistein and metal ions, which is also observed from the UV-vis studies. The changes in enthalpy (ΔH°) and entropy (ΔS°) of the interaction were found to be -14.64 kJ mol(-1) and +42.75 J mol(-1)K(-1) respectively. These values indicate the involvement of electrostatic interactions along with a hydrophobic association that results in a positive entropy change. CD analysis shows that there is a slight increase in the% α-helical content of BSA on binding with genistein at lower molar ratios. Warfarin and ibuprofen displacement studies in accordance with the molecular docking show that genistein binds to site I (subdomain IIA) of BSA.

Prolonged Glycation of Hen Egg White Lysozyme Generates Non Amyloidal Structures

Glycation causes severe damage to protein structure that could lead to amyloid formation in special cases. Here in this report, we have shown for the first time that hen egg white lysozyme (HEWL) does not undergo amyloid formation even after prolonged glycation in the presence of D-glucose, D-fructose and D-ribose. Cross-linked oligomers were formed in all the cases and ribose was found to be the most potent among the three sugars. Ribose mediated oligomers, however, exhibit Thioflavin T binding properties although microscopic images clearly show amorphous and globular morphology of the aggregates. Our study demonstrates that the structural damage of hen egg white lysozyme due to glycation generates unstructured aggregates.

Complex formation of rutin and quercetin with copper alters the mode of inhibition of Ribonuclease A

Rutin and quercetin, both minor components of green tea and their Cu(II) complexes interact with Ribonuclease A (RNase A) in a novel way. The effects of rutin, quercetin and their copper complexes on the catalytic activity of the protein were investigated. Rutin shows an enhancement in the ribonucleolytic activity whereas the copper complexes and quercetin behave as non‐competitive type inhibitors with Ki values in the μM range. The secondary structural changes of RNase A in presence of the ligands were measured by circular dichroism and Fourier transform infrared spectroscopy. The binding parameters were obtained using a fluorescence quenching analysis.

A simple assay for the ribonuclease activity of ribonucleases in the presence of ethidium bromide

The ribonuclease (RNase) activity of ribonucleases has been assayed by observing the change in fluorescence intensity of ethidium bromide on binding with yeast RNA. The binding of EtBr with RNA was monitored via UV-vis and fluorimetric methods. The degradation of RNA by RNase A was monitored by the change in fluorescence emission intensity of ethidium bromide at 600nm on excitation at 510nm. The ribonucleolytic activity of RNase A and angiogenin at various pH values was determined by this method. From this technique we have also determined the macroscopic pKa values of active site residues of these enzymes. This assay permits the evaluation of the catalytic efficiency of enzymatic proteins ranging from high ribonucleolytic activity to low ribonucleolytic activity toward the natural substrate RNA.

Binding of hen egg white lysozyme fibrils with nucleic acids.

Non proteinaceous substances are found to be associated with toxic protein aggregates commonly known as fibrils. Hen egg white lysozyme (HEWL) is able to form fibrillar species under various conditions. Here for the first time we report concentration dependent binding affinities of preformed HEWL fibrils towards DNA and RNA at physiological pH (pH 7.4). We have found that HEWL fibrils bind with DNA and RNA that is distinctly different when compared to native HEWL. The association constant (Ka) of native HEWL and ct-DNA at pH 7.4 is 6.8×10(5)M(-1). We have also investigated the conformational alterations of DNA that occur on binding with HEWL fibrils. Our study has demonstrated dominant electrostatic interactions between oppositely charged polyelectrolytes which accounts for the binding of nucleic acids with fibrils. The affinity between the moieties could lead to disruption in the functions of cellular components that might be attributed to the toxicity of the aggregates formed in vivo.

Location and binding mechanism of an ESIPT probe 3-hydroxy-2-naphthoic acid in unsaturated fatty acid bound serum albumins

The binding site and the binding mechanism of 3-hydroxy-2-naphthoic acid (3HNA) in oleic acid (OA) bound serum albumins (bovine serum albumin (BSA) and human serum albumin (HSA)) have been determined using steady state and time resolved emission of tryptophan residues (Trp) in proteins and the ESIPT emission of 3HNA. Time resolved anisotropy of the probe 3HNA and low temperature phosphorescence of Trp residues of BSA in OA bound BSA at 77K reveals a drastic change of the binding site of 3HNA in the ternary system compared to that in the free protein. 3HNA binds near Trp213 in the ternary system whereas 3HNA binds near Trp134 in the free protein. The structure of OA bound BSA generated using docking methodology exhibits U-bend configuration of all bound OA. The docked pose of 3HNA in the free protein and in OA bound albumins (ternary systems) and the concomitant perturbation of the structure of proteins around the binding region of 3HNA corroborate the enhanced ESIPT emission of 3HNA and the energy transfer efficiency from the donor Trp213 of BSA to 3HNA acceptor in 3HNA-OA-BSA system.

Effect of Temperature and Solvent on Fibrillation of Human Serum Albumin

During the past several years, studies on the protein aggregation process in the presence of cosolvents/ co-solutes have been looked into which provides significant insight in the stability of proteins in a crowded cellular milieu. Here, in the present report we have investigated the fibrillation of human serum albumin (HSA) under the mixed aqueous-ethanol solvent conditions at two different temperatures (37 °C and 65 °C). Self-association of protein was monitored using various spectroscopic and microscopic techniques. Results obtained from detailed investigation have shown that fibrillation of human serum albumin is favored at higher temperature (65 °C) at lower ethanol concentration. However, at 37 °C comparatively higher ethanol concentration is the prerequisite condition for fibrillation process to take place.

Comparison of the ribonucleolytic activity of the dityrosine cross-linked Ribonuclease A dimer with its monomer in the presence of inhibitors

Dityrosine cross-linking is considered to be one of the major reasons behind natural protein dimerization. Reports have shown that dimers of Ribonuclease A prepared by several methods exhibit reduced ribonucleolytic activity compared to the native monomer. Here in the present report, a detail investigation has been carried out to determine the effect of dityrosine cross-linking of Ribonuclease A on its ribonucleolytic activity. We have also studied the inhibitory property of natural, competitive and noncompetitive inhibitors on the catalytic activity of the dimer. The dimer was prepared by photo irradiation of Ribonuclease A using riboflavin as a photo sensitizer followed by separation using size exclusion chromatography. The dimer has been characterized via gel electrophoresis and various spectroscopic techniques. Experimental findings suggest that the synthesized dityrosine cross-linked dimer of Ribonuclease A exhibits reduced ribonucleolytic activity in comparison with the monomeric form both in the presence and absence of the inhibitors.

Interaction of (-)-Epigallocatechin Gallate with Lysozyme-Conjugated Silver Nanoparticles

The interaction of lysozyme (Lyz)-conjugated silver (Ag) nanoparticles with (–)-epigallocatechin gallate (EGCG), one of the major components of green tea, has been investigated. Interaction of a protein with ligand/drug molecules perturbs the conformation of secondary and tertiary structures of the protein. We have demonstrated the conformational changes in the tertiary structures of the Lyz molecules on EGCG binding using surface-enhanced Raman scattering (SERS) and circular dichroism (CD) spectroscopic measurements. From the analysis of the amide I band of Lyz in SERS and CD spectra, the site of interaction of EGCG with protein molecules in Lyz-conjugated Ag particles has been identified. Spectroscopic evidence for the conformational response of Trp62 and Trp63, in the β-domain of the protein, to the binding of EGCG has been discussed.

Interaction of (−)-Epigallocatechin Gallate and Silver Colloid with Bovine Serum Albumin:

The interaction between borate-ion-stabilized silver (Ag) nanoparticles and (–)-epigallocatechin gallate (EGCG) has been investigated. The added EGCG molecules chelate with the borate ions to form a borate ester. This complex provides a better stability to the metal particles by formation of a cap around them. The spectral line profile of the surface plasmon resonance band of the metal particles has been used to probe the metal–molecule interactions. The withdrawal of electron density from the surface of the Ag particle by the phenolic group of EGCG molecule results in a red-shift of the surface plasmon resonance band of the particles in the sol. In this study we show that both borate-ester-capped Ag nanoparticles and the borate ester itself can be transported by blood plasma serum albumin.