Hydrophobicity Is the Governing Factor in the Interaction of Human Serum Albumin with Bile Salts

The present study demonstrates a detailed characterization of the interaction of a series of bile salts, sodium deoxycholate (NaDC), sodium cholate (NaC), and sodium taurocholate (NaTC), with a model transport protein, human serum albumin (HSA). Here, steady-state and time-resolved fluorescence spectroscopic techniques have been used to characterize the interaction of the bile salts with HSA. The binding isotherms constructed from steady-state fluorescence intensity measurements demonstrate that the interaction of the bile salts with HSA can be characterized by three distinct regions, which were also successfully reproduced from the significant variation of the emission wavelength (λ_em) of the intrinsic tryptophan (Trp) moiety of HSA. The time-resolved fluorescence decay behavior of the Trp residue of HSA was also found to corroborate the steady-state results. The effect of interaction with the bile salts on the native conformation of the protein has been explored in a circular dichroism (CD) study, which reveals a decrease in α-helicity of HSA induced by the bile salts. In accordance with this, the esterase activity of the protein–bile salt aggregates is found to be reduced in comparison to that of the native protein. Our results exclusively highlight the fact that it is the hydrophobic character of the bile salt that governs the extent of interaction with the protein. Isothermal titration calorimetry (ITC) and molecular docking studies further substantiate our other experimental findings

Enhanced Binding of Phenosafranin to Triblock Copolymer F127 Induced by Sodium Dodecyl Sulfate: A Mixed Micellar System as an Efficient Drug Delivery Vehicle

In this study, we explored the interaction of a cationic phenazinium dye, phenosafranin (PSF, here used as a model drug), with pluronic block copolymer F127, both in the presence and in the absence of the anionic surfactant sodium dodecyl sulfate (SDS), which forms mixed micelles with F127. We applied both steady-state and time-resolved spectroscopic techniques, along with isothermal titration calorimetry (ITC), to demonstrate the binding of the probe PSF to both the pluronic and F127/SDS mixed micelles. Dynamic light scattering (DLS) study revealed that, upon interaction with SDS, the hydrodynamic diameter (<i>d</i>_H) of F127 micelles decreased due to the formation of the mixed micelles. The PSF penetrated to the more hydrophobic interior of the mixed micellar system as compared to F127 micelles alone. Micropolarity and fluorescence-quenching experiments revealed that PSF is more deeply seated in the case of F127/SDS mixed micelles. Through a partition coefficient, lifetime measurements, and time-resolved anisotropy experiments, we also established that the partitioning of the probe within the F127 micelles in the presence of SDS is almost double than that in its absence. ITC data corroborates the fact that the binding of PSF is the strongest and most thermodynamically favorable when mixed micelles are formed, which enables our system to serve as an excellent drug delivery vehicle when compared to F127 alone

Inverse Temperature Dependence in Static Quenching versus Calorimetric Exploration: Binding Interaction of Chloramphenicol to β‑Lactoglobulin

The binding interaction between the whey protein bovine β-lactoglobulin (βLG) with the well-known antibiotic chloramphenicol (Clp) is explored by monitoring the intrinsic fluorescence of βLG. Steady-state and time-resolved fluorescence spectral data reveal that quenching of βLG fluorescence proceeds through ground state complex formation, i.e., static quenching mechanism. However, the drug–protein binding constant is found to vary proportionately with temperature. This anomalous result is explained on the basis of the Arrhenius theory which states that the rate constant varies proportionally with temperature. Thermodynamic parameters like Δ<i>H</i>, Δ<i>S</i>, Δ<i>G</i>, and the stoichiometry for the binding interaction have been estimated by isothermal titration calorimetric (ITC) study. Thermodynamic data show that the binding phenomenon is mainly an entropy driven process suggesting the major role of hydrophobic interaction forces in the Clp−βLG binding. Constant pressure heat capacity change (Δ<i>C</i>_p) has been calculated from enthalpy of binding at different temperatures which reveals that hydrophobic interaction is the major operating force. The inverse temperature dependence in static quenching is however resolved from ITC data which show that the binding constant regularly decreases with increase in temperature. The modification of native protein conformation due to binding of drug has been monitored by circular dichroism (CD) spectroscopy. The probable binding location of Clp inside βLG is explored from AutoDock based blind docking simulation

Triblock-Copolymer-Assisted Mixed-Micelle Formation Results in the Refolding of Unfolded Protein

The present work reports a new strategy for triblock-copolymer-assisted refolding of sodium dodecyl sulfate (SDS)-induced unfolded serum protein human serum albumin (HSA) by mixed-micelle formation of SDS with poly­(ethylene oxide)-poly­(propylene oxide)-poly­(ethylene oxide) triblock copolymer EO₂₀PO₆₈EO₂₀ (P123) under physiological conditions. The steady-state and time-resolve fluorescence results show that the unfolding of HSA induced by SDS occurs in a stepwise manner through three different phases of binding of SDS, which is followed by a saturation of interaction. Interestingly, the addition of polymeric surfactant P123 to the unfolded protein results in the recovery of ∼87% of its α-helical structure, which was lost during SDS-induced unfolding. This is further corroborated by the return of the steady-state and time-resolved fluorescence decay parameters of the intrinsic tryptophan (Trp214) residue of HSA to the initial nativelike condition. The isothermal titration calorimetry (ITC) data also substantiates that there is almost no interaction between P123 and the native state of the protein. However, the mixed-micelle formation, accompanied by substantial binding affinities, removes the bound SDS molecules from the scaffolds of the unfolded state of the protein. On the basis of our experiments, we conclude that the formation of mixed micelles between SDS and P123 plays a pivotal role in refolding the protein back to its nativelike state

Contrasting Effects of Salt and Temperature on Niosome-Bound Norharmane: Direct Evidence for Positive Heat Capacity Change in the Niosome:β-Cyclodextrin Interaction

The modulation of the prototropic equilibrium of a cancer cell photosensitizer, norharmane (NHM), within a niosome microheterogeneous environment has been investigated. The contrasting effects of temperature and extrinsically added salt on the photophysics of niosome-bound drug have been meticulously explored from steady-state and time-resolved spectroscopic techniques. The cation ⇌ neutral prototropic equilibrium of NHM is found to be preferentially favored toward the neutral species with increasing salt concentration, and the results are rationalized on the basis of water penetration to the hydration layer of niosome. The effects are typically reversed with temperature. The differential rotational relaxation behavior of NHM under various conditions has also been addressed from fluorescence anisotropy decay. Further, the study delineates the application of β-cyclodextrin (βCD) as a potential host system, leading to drug sequestration from the niosome-encapsulated state. To this end, a detailed investigation of the thermodynamics of the niosome:βCD interaction has been undertaken by isothermal titration calorimetry (ITC) to unravel the notable dependence of the thermodynamic parameters on temperature. Consequently, a critical analysis of the variation of the enthalpy change (Δ<i>H</i>) of the process with temperature leads to the unique observation of a positive heat capacity change (ΔC_p) marking the hallmark of hydrophobic hydration

Temperature Induced Morphological Transitions from Native to Unfolded Aggregated States of Human Serum Albumin

The circulatory protein, human serum albumin (HSA), is known to have two melting point temperatures, 56 and 62 °C. In this present manuscript, we investigate the interaction of HSA with a synthesized bioactive molecule 3-pyrazolyl 2-pyrazoline (PZ). The sole tryptophan amino acid residue (Trp214) of HSA and PZ forms an excellent FRET pair and has been used to monitor the conformational dynamics in HSA as a function of temperature. Molecular docking studies reveal that the PZ binds to a site which is in the immediate vicinity of Trp214, and such data are also supported by time-resolved FRET studies. Steady-state and time-resolved anisotropy of PZ conclusively proved that the structural and morphological changes in HSA mainly occur beyond its first melting temperature. Although the protein undergoes thermal denaturation at elevated temperatures, the Trp214 gets buried inside the protein scaffolds; this fact has been substantiated by acrylamide quenching studies. Finally, we have used atomic force microscopy to establish that at around 70 °C, HSA undergoes self-assembly to form fibrillar structures. Such an observation may be attributed to the loss of α-helical content of the protein and a subsequent rise in β-sheet structure