Fabrication of a GUMBOS-based acid-base indicator: smart probe for sensing acids and bases in any solvent.

This report outlines the synthesis of an ionic liquid-based pH-responsive indicator to sense acids or bases in non-polar as well as polar solvents. Herein, we have assembled a new ionic liquid (IL) comprised of a group of uniform materials based on organic salts (GUMBOS) by attaching a quaternary phosphonium ionic liquid with a very common acid-base indicator, methyl orange, via simple ion-exchange reaction. This integrated IL-based indicator is highly soluble in less polar solvents and exhibits good sensitivity toward the presence of acids/bases in those media. Furthermore, this indicator has been exploited in determining the dissociation constants of several acids in non-aqueous aprotic solvents by overlapping indicator method and hence this report provides essential information toward the understanding of many fundamental chemical reactions. This report has further scope for the synthesis of novel aqueous suspended nanomaterials, i.e., the nanoparticles derived from GUMBOS (nanoGUMBOS) by a simple flash nano-precipitation method. The nanomaterial has been well characterized by different spectroscopic and microscopic studies. The obtained nanoparticles also exhibit substantial pH-responsive behaviors in aqueous medium and show better susceptibility as compared to the free organic indicator. Thus, this report explores detailed studies on the IL-based indicator in sensing the acidity/basicity of various media.

Effect of casein on pure lecithin liposome: Mixed biomacromolecular system for providing superior stabilization to hydrophobic molecules.

Formulation of new liposoma-based systems can always be in the spotlight for their unique utilization as carriers. Some changes in the composition of lipids may give rise to new mixed liposomes exhibiting modified size and physico-chemical characteristics. Consequently, these display different encapsulating properties toward various molecules. In this work, we have explored the variations in the model lecithin liposomes with casein additive from their size, shape, microenvironment and dynamics in solution. It is observed that with introduction of casein, the size of the liposome is substantially reduced due to incorporation of the additive in its bilayer. Strong interaction between the hydrophobic side chains of casein and lipid bilayer, and electrostatic repulsion of head groups of lipid are responsible to result small casein-mixed liposomes. Spectral properties of coumarin-153 disclose that the microenvironment of the bi-layer of mixed system is predominantly hydrophobic in nature and much rigid too. Fluorescence-lifetime-imaging-microscopy indicates that casein mixed systems exhibit wider lifetime distribution than pure liposomes with predominance of longer lifetimes, entirely arising from the outer bilayer. This indicates that casein gets incorporated in the bilayer of the vesicle to cater rigid and more hydrophobic microenvironment with an effective decrease in size. It is found that such mixed system is very efficient to stabilize the hydrophobic drugs (curcumin and ß-carotene) for prolonged period than the pure liposome or casein-only. The findings are indeed important from the perspective of drug stabilization by liposomes, and is suggestive of possible utilization in oral drug delivery applications involving such size-reduced nano-encapsulates.

A ZnS quantum dot-based super selective fluorescent chemosensor for soluble ppb-level total arsenic [As(iii) + As(v)] in aqueous media: direct assay utilizing aggregation-enhanced emission (AEE) for analytical application.

This study brings out a novel, superselective detection employing thiosalicylic acid-capped ZnS-based quantum dots that display photoluminescence "turn-on" characteristics only in the presence of arsenic in the aquatic medium for the first time. It shows a splendid limit of detection of soluble arsenic down to a few ppb level, much below than the MCL reported value, without being interfered by any other ions.

Simultaneous Binding of Folic Acid and Methotrexate to Human Serum Albumin: Insights into the Structural Changes of Protein and the Location and Competitive Displacement of Drugs.

Protein structure can be flexible to adopt multiple conformations to house small molecules. In this paper, we have attempted to experimentally figure out how the structure of a transport protein steer the drug-drug competition (DDC) by maintaining the equilibrium distribution of the bound and unbound fractions of drugs. This understanding is an important facet in biophysical and medicinal chemistry to ascertain the effectiveness of drugs. It is important to note that majority of studies involving small-molecule-transport protein interaction aimed at characterizing the binding process, and because these proteins can interact with thousands of molecules, there are hundreds of reports on such interactions. This ultimately led to an impression among the readers that any studies involving serum albumin may not lead to any new findings except for traditional binding explorations. However, in the present paper, we would like to draw the attention of the readers that our findings are very surprising, new, and important, involving the phenomenon of ligand-protein interaction. Here, we have studied two structurally similar drugs methotrexate (MTX) and folic acid (FA), which attempt to bind the primary binding site (subdomain IIA), one at a time, of human serum albumin. Details of binding analyses reveal that when both of the drugs are present, the single-site binding mode of FA prefers to occupy the primary binding site and hence pushes the primary-site-bound MTX to another location (subdomain IIIA), which is the second binding site of MTX. The structural analysis indicates that DDC has occurred in a cooperative fashion so that the loss of the protein secondary structure is minimum when both drugs are bound to the protein, which means that in the case of duo-drug binding, the conventional interaction between the drug and the protein is altered to undergo restoration of the protein structure. This can indeed regulate the DDC by modifying the bound and unbound fractions of MTX in plasma. The present study emphasizes that in vitro structural characterizations of the transport protein provide important information to improve the molecular-level understanding of DDC for further therapeutic applications with combination drug.

Investigations on the Effect of Fatty Acid Additives on Casein Micelles: Role of Ethylenic Unsaturation on the Interaction and Structural Diversity.

Casein, one of the major constituent of milk protein, is considered to be a good candidate for oral drug delivery system. Also, milk transports various essential fatty acid to blood through dietary supplements. In this study, we have explored the alteration in the structural characteristic in terms of the modulations in the microenvironment of the protein in the presence of different types of fatty acids. Herein, we have observed that the unsaturation of fatty acids mostly affects the structure of casein micelles (CMs) by impinging upon the hydrophobic force of interaction following a decrease in the electrostatic interaction of various amino acid unit. Alteration of such forces is responsible for the increase in the aggregate size, modification in the protein secondary structure, and different morphology of CMs. Fluorescence behavior of 4-(dicyanomethylene)-2-methyl-6-(4-dimethylaminostyryl)-4H-pyran indicates that the rigidity of the microenvironment is the main characteristic of the fatty acid binding, and the binding constant increases with the fatty acid chain length for saturated fatty acid or with the introduction of unsaturation onto it. Fluorescence lifetime imaging microscopy study indicates that the microstructure of CMs becomes more compact in the presence of unsaturated fatty acids, and this is also responsible for the increase in the diffusion time of the probe. Moreover, decrease in the fluorescence of extrinsic probe 8-anilinonaphthalene-1-sulfonate with the addition of unsaturated fatty acid reveals that these fatty acids alter the electrostatic interaction between casein units, more specifically in case of the surface-bound κ-casein. Therefore, this study provides a very useful information on the binding of fatty acids and helps to evaluate other fatty acid, as well as different small molecules binding in the applicative medicinal purpose.

Optical Spectroscopic and Morphological Characterizations of Curcuminized Silk Biomaterials: A Perspective from Drug Stabilization.

Silk protein fibroins have gained remarkable attention in recent years as a potential drug carrier in the developing medicinal field of research. In this work, the stability of anticancer agent curcumin in the presence of two different silk protein fibroins from nonmulberry Antheraea mylitta (Am) and mulberry Bombyx mori (Bm) has been examined, and the possible mechanism of stabilization in a physiologically relevant medium has also been explored. In solution phase, upon treatment with curcumin, the predominated ß-sheet structure of Am is marginally altered, whereas in the case of Bm, a substantial structural changeover has been observed (from coil to ß-sheet) to accommodate the hydrophobic drug. Also, the morphological assessments suggest that curcumin is nicely housed in the nanoscaffold of silk fibroin (SF). Consequently, the extent of degradation of curcumin is remarkably suppressed upon encapsulation with the SF. The dissimilarity in the binding patterns of curcumin with these silk proteins could be responsible for the observed difference in the stability orders. Curcumin binds the surface of Bm, whereas in Am, the drug is incorporated in the hydrophobic cavity, and as a consequence, the drug is effectively sequestered out of the aqueous medium. The increase in the fluorescence quantum yield upon interaction with the protein greatly modulates the excited-state intermolecular hydrogen atom transfer (ESIPT) process, which is in tune with a substantial increase in the lifetime of the excited-state of curcumin. The ESIPT is known to play a crucial role in the degradation of curcumin under physiological pH conditions, which perhaps implies its potential therapeutic activity in the presence of silk. The in-depth spectroscopic analyses of curcumin-SF complexes in aqueous medium can provide useful insights for further applicative developments in bioengineering.

Counterpointing Scenarios on the Fate of Different Prototropic Forms of Norfloxacin Housed in the Pocket of Lysozyme: The Nonelectrostatic Interactions in the Protein Interior Are in the Controlling Role on the Prototropic Equilibria of the Guest.

Herein, we report a comprehensive study on the interaction of three protomeric forms of the antibacterial drug norfloxacin (nfx) with the enzymatic protein human lysozyme (lyz). Norfloxacin, having the option for two-stage acid-base equilibria, converts from cationic (nfx+) to zwitterionic (nfx±) form, followed by an anionic (nfx-) species, with increasing pH. Among these protomeric forms, lysozyme binds nfx± most robustly, whereas nfx- has a weak association and nfx+ does not show any interaction. In lysozyme, the location of the drug was ascertained by competitive binding assay with 8-anilino-1-naphthalenesulfonate, and this was further examined with molecular docking simulation. The binding process was found to be primarily governed by hydrogen bonding and van der Waals interactions. The study has further revealed that preferential binding of nfx± by the protein over nfx- led to a switchover of nfx- to nfx±; and the resulting increased population of nfx± over the other is beneficial for the pharmacological activity of the drug in terms of its accumulation in the target bacterial cells. The present study accomplishes two important objectives. It holds significance regarding the differential interaction of multiprotomeric drugs with biomolecules, such as proteins, enzymes, lipid membranes, etc., and also on such biomolecule-assisted alteration of the acid-base equilibrium and consequent bioavailability of the drug. The findings are useful from the viewpoints of dispensation, distribution, and metabolism of any prototropic drug in living systems as they encounter several biomolecules in vivo. Another importance of this work stems from the study of comparative binding responses of lysozyme toward a drug existing in multiple forms depending on its protonation states or some other chemical processes.

Proton Transfer Pathways of 2,2'-Bipyridine-3,3'-diol in pH Responsive Fatty Acid Self-Assemblies: Multiwavelength Fluorescence Lifetime Imaging in a Single Vesicle.

Fatty acids are known to form different supramolecular aggregates in aqueous solutions depending on the pH of the medium. The dynamics of the transformation of oleate micelles into oleic acid/oleate vesicles has been investigated using a pH-sensitive intramolecular proton transfer fluorophore, 2,2'-bipyridine-3,3'-diol [BP(OH)2]. Different prototropic forms of BP(OH)2 exist in different pH values of the system, and thus, the ground state and the excited state dynamics of BP(OH)2 have been modulated in these confined media. The formation of different tautomeric forms of BP(OH)2 in oleate micelles (at basic pH) is confirmed using time-resolved emission spectra and fluorescence anisotropy measurements. The hydrophobic environment provided by these assemblies reduces the water-assisted nonradiative decay channels and lengthens the fluorescence lifetime of BP(OH)2. The rotational relaxation time in the micellar assembly is higher than that in the vesicle, which may be due to the higher microviscosity sensed by the fluorophore in the micelle. Besides, we have shown for the first time that BP(OH)2 can be used as a membrane-bound fluorophore, using fluorescence lifetime imaging microscopy (FLIM). A broad distribution in the size of the vesicle is observed from the FLIM image. Further, we have used multiwavelength FLIM to collect the FLIM images of a single vesicle at different emission wavelengths, and the lifetime distribution obtained from the FLIM images at different emission wavelengths in a single vesicle correlates well with the lifetime values obtained from the ensemble average measurements in the bulk solution.

Modulation and Salt-Induced Reverse Modulation of the Excited-State Proton-Transfer Process of Lysozymized Pyranine: The Contrasting Scenario of the Ground-State Acid-Base Equilibrium of the Photoacid.

Here we report on the excited-state behavior in terms of the excited-state proton-transfer (ESPT) reaction as well as the ground-state acid-base property of pyranine [8-hydroxypyrene-1,3,6-trisulfonate (HPTS)] in the presence of an enzymatic protein, human lysozyme (LYZ). HPTS forms a 1:1 ground-state complex with LYZ having the binding constant KBH = (1.4 ± 0.05) × 10(4) M(-1), and its acid-base equilibrium gets shifted toward the deprotonated conjugate base (RO(-)), resulting in a downward shift in pKa. This suggests that the conjugate base (RO(-)) is thermodynamically more favored over the protonated (ROH) species inside the lysozyme matrix, resulting in an increased population of the deprotonated form. However, for the release of the proton from the excited photoacid, interestingly, the rate of proton transfer gets slowed down due to the "slow" acceptor biological water molecules present in the immediate vicinity of the fluorophore binding region inside the protein. The observed ESPT time constants, ∼140 and ∼750 ps, of protein-bound pyranine are slower than in bulk aqueous media (∼100 ps, single exponential). The molecular docking study predicts that the most probable binding location of the fluorophore is in a region near to the active site of the protein. Here we also report on the effect of external electrolyte (NaCl) on the reverse modulation of ground-state prototropy as well as the ESPT process of the protein-bound pyranine. It is found that there is a dominant role of electrostatic forces in the HPTS-LYZ interaction process, because an increase in ionic strength by the addition of NaCl dislodges the fluorophore from the protein pocket to the bulk again. The study shows a considerably different perspective of the perturbation offered by the model macromolecular host used, unlike the available literature reports on the concerned photoacid.

Exploration of electrostatic interaction in the hydrophobic pocket of lysozyme: Importance of ligand-induced perturbation of the secondary structure on the mode of binding of exogenous ligand and possible consequences.

Exogenous ligand binding can be adequate to alter the secondary structure of biomolecules besides other external stimuli. In such cases, structural alterations can complicate on the nature of interaction with the exogenous molecules. In order to accommodate the exogenous ligand, the biomolecule has to unfold resulting in a considerable change to its properties. If the bound ligand can be unbound, the biomolecule gets the opportunity to refold back and return to its native state. Keeping this in mind, we have purposely investigated the interaction of tartrazine (TZ), a well abundant azo food colorant, with two homologous lysozymes, namely, human lysozyme (HLZ) and chicken egg white lysozyme (CEWLZ) in physiological pH condition. The binding of TZ with lysozymes has been identified to accompany a ligand-induced secondary structure alteration as indicated by the circular dichroism spectra, and the reduction of α-helical content is more with HLZ than CEWLZ. Interestingly, the binding is identified to occur in the electronic ground state of TZ with lysozyme in its hydrophobic cavity, containing excess of positive charge, predominantly via electrostatic interaction. With increase of salinity of the medium the protein tends to refold back due to wakening of electrostatic forces and consequent reduction of strength of ligand interaction and unbinding. The entropy enthalpy compensation (EEC) has been probed to understand the binding features and it is found that CEWLZ-TZ shows better compensation than HLZ-TZ complex. This is presumably due to the fact that with CEWLZ the binding does not accompany substantial change in the protein secondary structure and hence ineffective to scramble the EEC. The present study initiates the importance of ligand-perturbed structural alteration of biomolecule in controlling the thermodynamics of binding. If there is a considerable alteration of the protein secondary structure due to binding, it is indicative that such changes should bring in the overall loss of activity of protein.

Distilbene derivative as a new environment-sensitive bifunctional ligand for the possible induction of serum protein aggregation: a spectroscopic investigation and potential consequences.

The photophysical properties of a new distilbene fluorophore, DPDB, belonging to the conjugated polyene family is found to be well modulated with the variation of the microenvironment. Compared to the ground state, the excited-state photophysical properties of the fluorophore have been altered to larger extents with the variation of polarity and the hydrogen-bonding nature of solvents. The change in the fluorescence intensity of DPDB shows a nice correlation with the aggregation behavior of different surfactants which have been utilized for the determination of the CMC of surfactants. The distribution of DPDB is found to be higher in nonionic micelles. On the other hand, DPDB specifically binds the subdomain IB cavity of serum albumin with a stronger binding ability with HSA compared to BSA. DPDB behaves like a bivalent (bifunctional) ligand and forms a complex of 2:1 stoichiometry with serum albumins. Dynamic light scattering and circular dichroism measurements indicate that DPDB favors the association of serum albumin molecules, promoting their preaggregation state. Aggregation is an important phenomenon and is known to be initiated by heat, extreme pH conditions, very high ionic strength, surfactants, metal ions, and so forth. This study explores a new avenue in bringing about association phenomena of serum albumins and points out that the binding of such a bifunctional ligand may also become an important factor in inducing the protein association.

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.

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.

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 ß-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.

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.

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.

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.

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.

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.