Shubhashis Datta

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.

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.

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.

Exploration of pH-dependent behavior of the anion receptor pocket of subdomain IIA of HSA: determination of effective pocket charge using the Debye-Hückel limiting law.

Protein-ligand electrostatic interaction can be looked upon as ion receptor-ligand interaction, and the binding cavity of protein can be either an anion or cation receptor depending on the charge of the guest. Here we focus on the exploration of pH-modulated binding of a number of anionic ligands, specific to the subdomain IIA cavity of HSA, such as carmoisine, tartrazine, cochineal red, and warfarin. The logarithm of the binding constant is found to vary linearly with the square-root of ionic strength, indicating applicability of the Debye-Hückel limiting law to protein-ligand electrostatic binding equilibrium, and concludes that the subdomain IIA cavity is an anion receptor. The present approach is very unique that one can calculate the effective charge of the protein-based anion receptor pocket, and the calculated charge has been found to vary between +1 and +3 depending on the pH and ligand itself. The study also indicates that in such cases of specific ligand binding the pocket charge rather than the overall or surface charge of the macromolecule seems to have a paramount role in determining the strength of interaction. For the first time, it is demonstrated that the Debye-Hückel interionic interaction model can be successfully applied to understand the protein-based receptor-ligand electrostatic interaction in general.

Synthesis of a new class of furo[3,2-c]coumarins and its anticancer activity

A series of furo[3,2-c]coumarin derivatives 1a-d were synthesized and evaluated for their antiproliferative activity against MCF-7 breast and HCT-15 colon cancer cell lines using Sulfo-rhodamine B (SRB) assay. Compounds 1b and 1d showed higher antiproliferative activity than 1a and 1c. UV-Vis spectroscopy was used for DNA and BSA-binding affinity of the compounds 1b and 1d and gave overall affinity constants of K1b-DNA=8.1×10(3) M(-1), K1d-DNA=1.1×10(4) M(-1), K1b-BSA=5.1×10(4) M(-1), and K1d-BSA=7.6×10(4) M(-1). Our findings could provide new evidence showing the relationship between the chemical structure and anticancer activity of these new coumarin analogs.

Detailed scenario of the acid-base behavior of prototropic molecules in the subdomain-IIA pocket of serum albumin: results and prospects in drug delivery.

The protein pocket performs magically in controlling, inhibiting, or optimizing various biochemical processes. The elegant 3D disposition of different side chains in the cavity is a key point in accommodating specific ligands. Anion receptors in the subdomain-IIA pocket of serum albumin (SA) prefer to home anionic ligands. Acid-base behavior is an important property that relates to bioavailability and action of prototropic molecules/drugs. The present study provides a comprehensive understanding of the effect of subdomain-IIA pocket-specific interaction on the acid-base equilibrium of housed guests. The pKa of subdomain-IIA binder basic drugs decreases due to unfavorable interaction with the cationic drug species, while the decrease in the pKa of acidic drugs is due to favored binding of the deprotonated species presumably via electrostatic interaction with anion receptors. Acidity-shifting efficacy of albumins is introduced for the first time using the pKa-shifting index (α), a unique parameter for a given prototropic-drug-host pair to assess bioavailability. The acidic drug warfarin and the basic drug fuberidazole, showing a high α-value, should be efficient in drug-SA cocktail, and those with low α should be less efficient. Use of the pKa-shifting index for prototropy-based drugs should enable the drug efficacy to be evaluated smartly for similar systems. Shifting of the pKa of protein-encapsulated drugs stems the possibility of albumin-based delivery systems for extracting the therapeutically active species.

Reorganization energy and Stokes shift calculations from spectral data as new efficient approaches in distinguishing the end point of micellization/aggregation

Critical micellar concentration (CMC) is an important parameter which indicates the matured associated state of amphiphilic molecules. The present work demonstrates a new, accurate, and generalized method for the determination of the critical micellar concentration (CMC) based on the estimation of the reorganization energy (RE) and Stokes shift of fluorescent probes placed inside micellar aggregates. With increasing concentration of surfactant, the fluorophore is well portioned in the micellar environment. The change in the magnitude of the Stokes shift and reorganization energy with respect to bulk water is a result of the formation of aggregates from surfactant monomers and there is a substantial change of RE in the vicinity of the CMC like what is observed with various other physical parameters during micellization. CMC values determined from both Stokes shift calculation and reorganization energy estimations are comparable with those previously reported in the literature. However, we demonstrate that reorganization energy calculation provides improved consistency and is more user-friendly than that estimated from Stokes shift measurements.

Phosphorescence Kinetics of Singlet Oxygen Produced by Photosensitization in Spherical Nanoparticles. Part I. Theory

The singlet oxygen produced by energy transfer between an excited photosensitizer (pts) and ground-state oxygen molecules plays a key role in photodynamic therapy. Different nanocarrier systems are extensively studied to promote targeted pts delivery in a host body. The phosphorescence kinetics of the singlet oxygen produced by the short laser pulse photosensitization of pts inside nanoparticles is influenced by singlet oxygen diffusion from the particles to the surrounding medium. Two theoretical models are presented in this work: a more complex numerical one and a simple analytical one. Both the models predict the time course of singlet oxygen concentration inside and outside of the spherical particles following short-pulse excitation of pts. On the basis of the comparison of the numerical and analytical results, a semiempirical analytical formula is derived to calculate the characteristic diffusion time of singlet oxygen from the nanoparticles to the surrounding solvent. The phosphorescence intensity of singlet oxygen produced in pts-loaded nanocarrier systems can be calculated as a linear combination of the two concentrations (inside and outside the particles), taking the different phosphorescence emission rate constants into account.