Mintu Halder

Optical Spectroscopic Exploration of Binding of Cochineal Red A with Two Homologous Serum Albumins

Cochineal Red A is a negatively charged synthetic azo food colorant and a potential carcinogen. We present here the study of binding of Cochineal Red A with two homologous serum albumins, human (HSA) and bovine (BSA), in aqueous pH 7.4 buffer by optical spectroscopic techniques. Protein intrinsic fluorescence quenching by Cochineal Red A occurs through ground-state static interaction and its binding with BSA is stronger than with HSA. The magnitudes of thermodynamic parameters suggest that dye binding occurs principally via electrostatic complexation. Site-marker competitive binding shows that Cochineal Red A binds primarily to site I of serum albumins. Circular dichroic spectra indicate that dye binding results in some conformational modification of serum albumins. Increased ionic strength of the medium results in lowering of binding. This study provides an important insight into possible means of removal of dye toxicity.

Spectroscopic Investigation of the Effect of Salt on Binding of Tartrazine with Two Homologous Serum Albumins: Quantification by Use of the Debye–Hückel Limiting Law and Observation of Enthalpy–Entropy Compensation

Formation of ion pair between charged molecule and protein can lead to interesting biochemical phenomena. We report the evolution of thermodynamics of the binding of tartrazine, a negatively charged azo colorant, and serum albumins with salt. The dye binds predominantly electrostatically in low buffer strengths; however, on increasing salt concentration, affinity decreases considerably. The calculated thermodynamic parameters in high salt indicate manifestation of nonelectrostatic interactions, namely, van der Waals force and hydrogen bonding. Site-marker competitive binding studies and docking simulations indicate that the dye binds with HSA in the warfarin site and with BSA at the interface of warfarin and ibuprofen binding sites. The docked poses indicate nearby amino acid positive side chains, which are possibly responsible for electrostatic interaction. Using the Debye-Hückel interionic attraction theory for binding equilibria, it is shown that, for electrostatic binding the calculated free energy change increases linearly with square root of ionic strength. Also UV-vis, fluorescence, CD data indicate a decrease of interaction with salt concentration. This study quantitatively relates how ionic strength modulates the strength of the protein-ligand electrostatic interaction. The binding enthalpy and entropy have been found to compensate one another. The enthalpy-entropy compensation (EEC), general property of weak intermolecular interactions, has been discussed.

Modulation of accessibility of subdomain IB in the pH-dependent interaction of bovine serum albumin with Cochineal Red A: a combined view from spectroscopy and docking simulations.

Our recent report on the binding of Cochineal Red A, a food dye, with HSA and BSA at pH 7.4 has revealed that electrostatic forces is the principal cause of interaction. In that study issues relating to complications arising out of modulation of dye binding affinity of BSA with pH had not been explored. Here we have further explored the interaction of Cochineal Red A with BSA in pH range 4.8-7.8. Surprisingly, this system behaves differently in the texture of interaction pattern at two extremes of studied pH range, unlike HSA. Importantly, the charge on the amino acid side chains in the binding pocket is likely to play a significant role.

Generation of Fluorescent Adducts of Malondialdehyde and Amino Acids: Toward an Understanding of Lipofuscin¶

Lipofuscin is a yellow‐brown, highly fluorescent pigment that undergoes an age‐related progressive accumulation in animal cells, mainly in postmitotic cells. It is a heterogeneous, high‐molecular weight material associated with proteins, lipids and nucleic acids. Lipofuscin is implicated in many aspects of human health, including aging, oxidative stress, macular degeneration, lipid peroxidation, atherosclerosis, dementia (Alzheimers Disease) and diseases associated with prions. Although the fluorescent properties of lipofuscin have long been recognized, neither histologists nor chemists have yet isolated the pigments themselves or characterized their optical properties. We have prepared lipofuscinlike species by reacting malondialdehyde (MDA) with cysteine (Cys). MDA: Cys adducts 3:2 and 2:2 are two of those that have been identified among the many that were present in the reaction. Whereas previous attempts to synthesize lipofuscinlike species resulted in compounds that were either nonfluorescent or emitted principally in the blue, the MDA: Cys adducts reported in this study are not only fluorescent but also emit over a broader range.

pH-insensitive electrostatic interaction of carmoisine with two serum proteins: A possible caution on its uses in food and pharmaceutical industry

Here we have investigated the binding of carmoisine, a water-soluble azo food colorant, with serum proteins (HSA and BSA) by fluorescence and UV-VIS spectroscopy, circular dichroism and molecular docking studies. Results indicate that fluorescence quenching of protein has been due to site-specific binding of the dye with biomacromolecules. Site marker competitive binding and molecular docking explorations show that interaction occurs in the sub-domain ІІA of HSA and the sub-domains ІІA and ІB in the case of BSA. Conformational investigation indicates that dye binding modifies the secondary structure of proteins and this also alters the microenvironment of the tryptophan(s). The interaction is found to be pH-insensitive which can have relevance to the toxicological profiles of the dye, and ionic strength dependence of binding can be exploited in protein purification mediated by such food colorants.

A single source-precursor route for the one-pot synthesis of highly luminescent CdS quantum dots as ultra-sensitive and selective photoluminescence sensor for Co2+ and Ni2+ ions

In this study, we have demonstrated a facile, simple one-pot and low cost method for the synthesis of 3-mercaptopropionic acid (MPA)-capped, water-soluble CdS quantum dots (QDs) with highly tunable optical properties. Initially Cd2+ coordinates with MPA at about pH 5, and the CdS QDs were then formed at a higher pH (7–12) under refluxing conditions through the disruption of coordination interaction with the release of sulfur. Here MPA played a dual role, as both, a source of sulfur and as a stabilizer. The particle size and the optical properties of the as-prepared CdS QDs were found to be dependent on the refluxing time for a given concentration ratio of the reactants and pH of the initial mixture. The broadness and large Stokes shift of emission of MPA–CdS QDs are due to the surface-trap state photoluminescence (PL). The PL peak around 510 nm–650 nm is due to the recombination of shallow trapped electrons in sulfur vacancy defect states with holes in the valence band, and a ∼665 nm peak (shoulder) arises from deep-trap states. The origin of the longer lifetime is presumed to be due to the involvement of surface-trap states and their environment. Use of MPA as a capping agent eventually enhances the water solubility as well as the stability of CdS QDs, which makes them useful for the ultra-sensitive detection of Co2+ and Ni2+. The selective coordination interaction of Co2+ and Ni2+ with MPA–CdS QDs through the carboxyl group of MPA provides a turn-off photoluminescence-based assay for sensitive detection of these metal ions without any interference of other commonly coexisting metal ions. The limit of detection (LOD) is 10 nM for Co2+ ions and 50 nM for Ni2+ ions. Co2+-induced color (from colorless to yellow) and UV-vis spectral change of MPA–CdS QDs is the simple way to distinguish Co2+ from Ni2+ in a higher concentration range (more than 5 µM). On the other hand the lower stability of the Co(II)–MPA complex than the Ni(II)–MPA complex provides a disodium salt of ethylenediaminetetraacetic acid (EDTA)-induced, time dependent turn-on photoluminescence-based technique to distinguish Co2+ from Ni2+ in the entire range of concentrations. EDTA-induced time dependent PL recovery of MPA–CdS QDs occurs via rapid dissociation of Co2+ ions from the surface of QDs than that of Ni2+. Thus our synthesized MPA–CdS QDs offer a very simple, rapid, cost-effective, turn-off–on photoluminescence-based technique for ultra-sensitive and selective detection of either Co2+ or Ni2+ in aqueous solution without interference of other common metal ions.

Accumulation and Interaction of Hypericin in Low‐density Lipoprotein— A Photophysical Study

The accumulation and interaction of hypericin with the biologically important macromolecule, low‐density lipoprotein (LDL), is investigated using various steady‐state and time‐resolved fluorescence measurements. It is concluded that multiple hypericins can penetrate considerably deeply into the LDL molecule. Up to ∼20 nonaggregated hypericin molecules can enter LDL; but upon increasing the hypericin concentration, the fluorescence lifetime of hypericin decreases drastically, suggesting most likely the self‐quenching of aggregated hypericin. There is also evidence of energy transfer from tryptophans of the constituent protein, apoB‐100, to hypericin in LDL. The results demonstrate the ability of LDL to solubilize hypericin (a known photosensitizer) in nonaggregated form, which has implications for the construction of drug delivery systems.

Effect of encapsulation in the anion receptor pocket of sub-domain IIA of human serum albumin on the modulation of pKa of warfarin and structurally similar acidic guests: a possible implication on biological activity.

Supramolecular and bio-supramolecular host assisted pKa shift of biologically relevant acidic guests, warfarin and coumarin 343, has been monitored using both steady-state and time resolved fluorescence spectroscopy. The anion receptors present in sub-domain IIA of human serum albumin (HSA) stabilize the anionic form of the guest and thereby shift pKa towards acidic range. On the other hand, the preferential binding of the neutral form of guests in the non-polar hydrophobic cavity of β-cyclodextrin results in up-shifted pKa. This shifting of pKa of drugs like warfarin, etc., whose therapeutic activity depends on the position of the acid-base equilibrium in human system, is of great importance in pharmacokinetics. The release of the active form of such drugs from macrocyclic carrier and subsequent distribution through the carrier protein should depend on the modulation of the overall pKa window brought about by the encapsulation in these hosts. Present work also suggests that properly optimized encapsulation in appropriate receptor pocket can enhance the bioavailability of drugs. This work also opens up the possibility to use HSA as encapsulator, instead of traditional cyclodextrins or other polymeric hosts, since such system may overcome toxicity as well as biocompatibility issues.

Detailed scrutiny of the anion receptor pocket in subdomain IIA of serum proteins toward individual response to specific ligands: HSA-pocket resembles flexible biological slide-wrench unlike BSA.

Present study reveals that the subdomain IIA cavity of two homologous serum albumins (HSA, BSA) has inherent mutual structural and functional deviations which render noticeable difference in behavior toward specific ligands. The major drug binding site (subdomain IIA) of HSA is found to be largely hydrophobic while that of BSA is partially exposed to water. Larger shift in REE spectra and greater change in solvent reorganization energy of coumarin 343 (C343)-anion in HSA clearly reveals that binding pocket is relatively large and water molecules penetrate deeper into it unlike BSA. The individual response of proteins to perturbation by ligands is found to be way different. Although the subdomain IIA is primarily anion receptive (prefers anionic ligands), the present study suggests that HSA may also like to bind neutral guests due to its remarkable conformational features. Actually, HSA is capable of adopting favorable conformation like mechanical slide-wrench, when required, to accommodate neutral ligands [e.g., coumarin 314 (C314)], as well. But due to less flexible solution structure, BSA behaves like fixed mechanical spanners and hence is not very responsive to C314. Therefore, the generally speaking functional-structural similarities of homologous proteins can be apparent and needs to be analyzed exhaustively.

Solvation Dynamics in Protein Environments: Comparison of Fluorescence Upconversion Measurements of Coumarin 153 in Monomeric Hemeproteins with Molecular Dynamics Simulations

The complexes of the fluorescence probe coumarin 153 with apomyoglobin and apoleghemoglobin are used as model systems to study solvation dynamics in proteins. Time-resolved Stokes shift experiments are compared with molecular dynamics simulations, and very good agreement is obtained. The solvation of the coumarin probe is very rapid with approximately 60% occurring within 300 fs and is attributed to interactions with water (or possibly to the protein itself). Differences in the solvation relaxation (or correlation) function C(t) for the two proteins are attributed to differences in their hemepockets.