Interaction of Lysozyme with Monocationic and Dicationic Ionic Liquids: Toward Finding a Suitable Medium for Biomacromolecules.

With an objective to understand the differences in the behavior of monocationic and dicationic ionic liquids (ILs) in their interaction with protein, we have investigated the binding interaction of lysozyme enzyme with two monocation ionic liquids (MILs), [C3MIm][Br], [C6MIm][Br], and one dicationic ionic liquid (DIL), [C6(MIm)2][Br]2, by exploiting various experimental methods. These ILs are purposefully chosen so that the effect of both hydrophobicity and structural arrangements of the cationic moiety of ionic liquids (ILs), if any, on the interaction event is understood. Both average ensemble and single molecule pathways have been adopted to obtain a comprehensive picture. For ensemble averaged measurements, the interaction events have been investigated by steady-state and time-resolved fluorescence spectroscopy, whereas for single molecule measurements, fluorescence correlation spectroscopy (FCS) has been utilized. Additionally, the behavior of protein in the absence and presence of ILs has also been investigated through circular dichroism (CD) measurements. The investigations have revealed that MILs and DIL interact differently with the protein. In particular, as compared to MILs, the influence of DIL toward protein is observed to be significantly less in terms of change in the structure and dynamics of protein. The outcome of the present work has demonstrated that imidazolium-based DIL can be a better choice over MILs for retaining native structure of protein in aqueous medium.

Probing the Interactions of 1-Alkyl-3-methylimidazolium Tetrafluoroborate (Alkyl = Octyl, Hexyl, Butyl, and Ethyl) Ionic Liquids with Bovine Serum Albumin: An Alkyl Chain Length-Dependent Study.

Herein, we have investigated the binding interaction of bovine serum albumin (BSA) with a series of 1-alkyl-3-methylimidazolium tetrafluoroborate (alkyl = ethyl, butyl, hexyl, and octyl) ionic liquids (ILs) in physiological buffer medium. The ILs are chosen basically to understand the effect of alkyl chain length on IL-protein interaction. Experiments have shown that the quenching of fluorescence of BSA is induced by relatively longer alkyl chain-containing ILs, [OMIM][BF4] and [HMIM][BF4]. The enthalpy-driven spontaneous binding (-ve Δ G) of hexyl and octyl chain-containing ILs with the protein is mediated by both hydrogen-bonding and van der Waals interactions. The experimental data have categorically explained the denaturation of protein conformation upon interaction with both [OMIM][BF4] and [HMIM][BF4]. The molecular docking calculation nicely corroborates the experimentally obtained results. The present study reveals that neither a smaller alkyl group-containing IL nor a very large alkyl group-containing IL is necessary to have effective protein-IL interactions. The study also reveals the influence of hydrophobic interaction over and above the hydrogen-bonding interaction on protein-IL binding events and essentially gives an idea about the optimum hydrophobic character of the ILs that is necessary to induce protein-IL interaction and consequently the denaturation of the protein structure.

Correlation of cholinergic drug induced quenching of acetylcholinesterase bound thioflavin-T fluorescence with their inhibition activity.

The development of new acetylcholinesterase inhibitors (AChEIs) and subsequent assay of their inhibition efficiency is considered to be a key step for AD treatment. The fluorescence intensity of thioflavin-T (ThT) bound in the active site of acetylcholinesterase (AChE) quenches substantially in presence of standard AChEI drugs due to the dynamic replacement of the fluorophore from the AChE active site as confirmed from steady state emission as well as time-resolved fluorescence anisotropy measurement and molecular dynamics simulation in conjunction with docking calculation. The parametrized % quenching data for individual system shows excellent correlation with enzyme inhibition activity measured independently by standard Ellman AChE assay method in a high throughput plate reader system. The results are encouraging towards design of a fluorescence intensity based AChE inhibition assay method and may provide a better toolset to rapidly evaluate as well as develop newer AChE-inhibitors for AD treatment.

Lysozyme binding ability toward psychoactive stimulant drugs: Modulatory effect of colloidal metal nanoparticles.

The interaction and binding behavior of the well-known psychoactive stimulant drugs theophylline (THP) and theobromine (THB) with lysozyme (LYS) was monitored by in-vitro fluorescence titration and molecular docking calculations under physiological condition. The quenching of protein fluorescence on addition of the drugs is due to the formation of protein-drug complex in the ground state in both the cases. However, the binding interaction is almost three orders of magnitude stronger in THP, which involves mostly hydrogen bonding interaction in comparison with THB where hydrophobic binding plays the predominant role. The mechanism of fluorescence quenching (static type) remains same also in presence of gold and silver nanoparticles (NPs); however, the binding capacity of LYS with the drugs changes drastically in comparison with that in aqueous buffer medium. While the binding affinity of LYS to THB increases ca. 100 times in presence of both the NPs, it is seen to decrease drastically (by almost 1000 fold) for THP. This significant modulation in binding behavior indicates that the drug transportation capacity of LYS can be controlled significantly with the formation protein-NP noncovalent assembly system as an efficient delivery channel.

Fluorescence properties and sequestration of peripheral anionic site specific ligands in bile acid hosts: Effect on acetylcholinesterase inhibition activity.

The increase in fluorescence intensity of model acetyl cholinesterase (AChE) inhibitors like propidium iodide (PI) and ethidium bromide (EB) is due to sequestration of the probes in primary micellar aggregates of bile acid (BA) host medium with moderate binding affinity of ca. 10(2)-10(3)M(-1). Multiple regression analysis of solvent dependent fluorescence behavior of PI indicates the decrease in total nonradiative decay rate due to partial shielding of the probe from hydrogen bond donation ability of the aqueous medium in bile acid bound fraction. Both PI and EB affects AChE activity through mixed inhibition and consistent with one site binding model; however, PI (IC50=20±1µM) shows greater inhibition in comparison with EB (IC50=40±3µM) possibly due to stronger interaction with enzyme active site. The potency of AChE inhibition for both the compounds is drastically reduced in the presence of bile acid due to the formation of BA-inhibitor complex and subsequent reduction of active inhibitor fraction in the medium. Although the inhibition mechanism still remains the same, the course of catalytic reaction critically depends on equilibrium binding among several species present in the solution; particularly at low inhibitor concentration. All the kinetic parameters for enzyme inhibition reaction are nicely correlated with the association constant for BA-inhibitor complex formation.

Human serum albumin reduces the potency of acetylcholinesterase inhibitor based drugs for Alzheimer's disease.

Human serum albumin (HSA) induced modulation of acetylcholinesterase (AChE) inhibition activity of four well-known cholinergic inhibitors like tacrine hydrochloride (TAC), donepezil hydrochloride monohydrate (DON), (-) Huperzine A (HuPA), eserine (ESE) was monitored quantitatively by Ellman's method. Kinetic analysis of enzyme hydrolysis reaction revealed that while the mechanism of inhibition does not change significantly, the inhibition efficiency changes drastically in presence of HSA, particularly for DON and TAC. However, interestingly, no notable difference was observed in the cases of HuPA and/or ESE. For example, the IC50 value of AChE inhibition increases by almost 135% in presence of ∼250 µM HSA (IC50 = 159 ± 8 nM) while comparing with aqueous buffer solution of pH 8.0 (IC50 = 68 ± 4 nM) in DON. On the other hand, the change is almost insignificant (<10%) in case of HuPA under the similar condition. The experimentally observed difference in the extent of modulatory effect was correlated with the sequestration ability of HSA towards different drugs predicted from molecular docking calculations. The result in this study demonstrates the importance to consider the plasma protein binding tendency of a newly synthesized AD drug before claiming its potency over the existing one. Further, development of new and intelligent delivery medium that shields the administered drugs from serum adsorption may reduce the optimal drug dose requirement.

Caffeine and sulfadiazine interact differently with human serum albumin: A combined fluorescence and molecular docking study.

The interaction and binding behavior of the well-known drug sulfadiazine (SDZ) and psychoactive stimulant caffeine (CAF) with human serum albumin (HSA) was monitored by in vitro fluorescence titration and molecular docking calculations under physiological condition. The quenching of protein fluorescence on addition of CAF is due to the formation of protein-drug complex in the ground state; whereas in case of SDZ, the experimental results were explained on the basis of sphere of action model. Although both these compounds bind preferentially in Sudlow's site 1 of the protein, the association constant is approximately two fold higher in case of SDZ (∼4.0×10(4)M(-1)) in comparison with CAF (∼9.3×10(2)M(-1)) and correlates well with physico-chemical properties like pKa and lipophilicity of the drugs. Temperature dependent fluorescence study reveals that both SDZ and CAF bind spontaneously with HSA. However, the binding of SDZ with the protein is mainly governed by the hydrophobic forces in contrast with that of CAF; where, the interaction is best explained in terms of electrostatic mechanism. Molecular docking calculation predicts the binding of these drugs in different location of sub-domain IIA in the protein structure.

Interaction of Sulfadiazine with Model Water Soluble Proteins: A Combined Fluorescence Spectroscopic and Molecular Modeling Approach.

The binding behavior of antibacterial drug sulfadiazine (SDZ) with water soluble globular proteins like bovine as well as human serum albumin (BSA and HSA, respectively) and lysozyme (LYS) was monitored by fluorescence titration and molecular docking calculations. The experimental data reveal that the quenching of the intrinsic protein fluorescence in presence of SDZ is due to the strong interaction in the drug binding site of the respective proteins. The Stern-Volmer plot shows positive deviation at higher quencher concentration for all the proteins and was explained in terms of a sphere of action model. The calculated fluorophore-quencher distances vary within 4 ~ 11 Å in different cases. Fluorescence experiments at different temperature indicate thermodynamically favorable binding of SDZ with the proteins with apparently strong association constant (~10(4)-10(5) M(-1)) and negative free energy of interaction within the range of -26.0 ~ -36.8 kJ mol(-1). The experimental findings are in good agreement with the respective parameters obtained from best energy ranked molecular docking calculation results of SDZ with all the three proteins.

Fluorescence modulation and associative behavior of lumazine in hydrophobic domain of micelles and bovine serum albumin.

The photophysical behavior of the deprotonated form of lumazine (Lum-anion) was studied in biologically relevant surfactant systems like sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB) and TritonX-100 (TX-100) and also model water soluble protein, bovine serum albumin (BSA), using steady-state and time-resolved fluorescence spectroscopy in buffer solution of pH 12.0. The association constant values were calculated from modulated fluorescence intensity of Lum-anion in different medium. The interaction of non-ionic surfactant TX-100 was found to be about 10 times greater than SDS and CTAB. However, while the driving force of binding in SDS and/or TX-100 is mainly hydrophobic in nature, electrostatic interaction with the oppositely charged micellar head group is the predominant factor in CTAB. The thermodynamic parameters like enthalpy (ΔH) and entropy (ΔS) change, etc., corresponding to the binding of Lum-anion with BSA were estimated by performing the fluorescence titration experiment at different temperatures. Thermodynamically favorable and strong binding of Lum-anion (K~10(4) M(-1)) into BSA is due to hydrophobic interaction in the ligand binding domain II. However, the binding mechanism is entirely different in presence of protein denaturing agent like urea and electrostatic interaction plays a major role under this condition.