Resonance Energy Transfer Theory Research Articles

Anticancer drug design of potassium ion (K+) assisted self-assembled Cu(II) Schiff base complex and its Ni(II) and Zn(II) analogues (3d-4s): A comparative study of DNA/protein (BSA) interactions and cytotoxicity

In the research for the therapeutic agents, three novel dinuclear 3d-4s complexes with a glycine-derived Schiff base ligand and a side-on bridging azido (µ-1,3-N3) ligand were synthesized and characterized, with formulas [K(CuL)(µ-1,3-N3)] (KCuL), [K(NiL)(µ-1,3-N3)] (KNiL) and [K(ZnL)(µ-1,3-N3)] (KZnL), where L indicates the Schiff base containing stoichiometric ratio of glycine and salicylaldehyde. Different structural, spectral and analytical methods, such as single-crystal X-ray crystallography, CHN analysis, FT-IR, UV–Vis and mass spectroscopy, were used for the characterization. The single-crystal X-ray diffraction of the KCuL complex confirmed the presence of a dinuclear complex containing one Cu(II) and K+centre, one Schiff base ligand and one side-on exogenous bridging azido ligand (μ-1,3-N3) in the asymmetric unit. A considerable anticancer activity of the complexes was explored on different cancerous cell lines such as breast, lung carcinoma, gastric and enocarcinoma and normal fibroblasts. DNA cleavage experiment showed that all the complexes cleave pGFPC1 supercoiled DNA in the presence of an activator like H2O2. The interaction between the complexes and Fish Sperm DNA (FS-DNA) was inspected by UV–Vis and fluorescence titration spectrometry, viscosity measurement, cyclic voltammetry and docking studies, with intercalation found as the main binding mode. The order of intercalation propensity of the complexes was deduced from Kb values as KCuL > KNiL > KZnL, and this order was confirmed by the competitive EtBr/complex binding experiment. Absorption and fluorescence spectroscopy revealed a static quenching mechanism as the interaction mechanism for binding between the complexes and bovine serum albumin protein (BSA). Synchronous fluorescence and site-marker competitive experiments completed the investigations. Also, Forster resonance energy transfer theory (FRET) was employed to calculate the distance, r, between the acceptor (complex) and the donor (BSA). The quantitative tendency of the complexes to interact with BSA was calculated as KSV, which varied in the order KCuL > KNiL > KZnL. Besides the results from the experimental methods, the theoretical molecular docking study and the Hirshfeld surface study alongside finger print plots were applied to confirm the mode and site of binding of the KCuL complex with DNA and BSA.

Tailoring spectroscopic properties of 1,10-phenanthroline monohydrate with TiO2 NPs: Unravelling thermodynamic parameters, FRET dynamics, and solvatochromic behavior

This study delves into the interaction between Titanium dioxide nanoparticles (TiO2 NPs) and 1,10-Phenanthroline Monohydrate (1,10-Phen), with a focus on quenching mechanisms and thermodynamic properties employing spectroscopic tools. Analysis reveals a decrease in quenching constants (KSV) at elevated temperatures (298 K, 303 K, 308 K), indicating static quenching mechanisms. Consistent binding sites (∼3) across temperatures suggest the formation of ground state complexes, supported by negative Gibbs free energy (ΔGo) values, confirming spontaneous complexation. The interaction between 1,10-Phen and TiO2 NPs manifests predominantly as electrostatic and ionic, inferred from the negative change in enthalpy (ΔHo) and positive change in entropy (ΔSo) values. Utilizing Foster resonance energy transfer theory (FRET), the distance between acceptor and donor is <70 Å, suggests significant energy transfer. Solvatochromic study helps in understanding the dipole interaction with the fluorophore. This research holds promise for biomedical applications, particularly in pharmaceutical contexts.

The interaction between resveratrol and catalase was studied by multispectral method and molecular docking simulation

In this research, we delved into the interaction mechanism between Resveratrol (RES) and Catalase (CAT) utilizing a suite of advanced analytical techniques, including multispectral analysis and molecular docking simulation. A comprehensive assessment of their binding characteristics, modalities, distances, and the subsequent impact on CAT's secondary structure was conducted through fluorescence spectroscopy, ultraviolet spectroscopy, circular dichroism, three-dimensional fluorescence spectroscopy, synchronous fluorescence spectroscopy, and molecular docking simulation. By employing the Stern-Volmer equation to determine KSV and Kq values, Our findings indicate that the RES-CAT interaction results in static fluorescence quenching, By conducting experiments, the binding constant KA and the number of binding sites n for CAT and RES were determined, with the number of binding sites n being close to 1, indicating the presence of a specific binding site between CAT and RES. Parameters at 298 K. The negative values of ΔH and ΔS suggest that van der Waals forces and hydrogen bonding are predominant in mediating the interaction between CAT and RES. The negativity of ΔG indicates the spontaneity of the reaction. The Fӧrster resonance energy transfer theory calculation revealed a binding distance of 4.917 nm, substantiating the occurrence of non-radiative energy transfer between RES and CAT. Furthermore, the UV-VIS absorption spectrum, synchronous fluorescence spectrum, three-dimensional fluorescence spectrum, and circular dichroism analyses collectively demonstrated that RES induces alterations in CAT's secondary structure, augmenting the polarity around tryptophan (Trp) residues, reducing hydrophobic interactions, and bolstering hydrophilicity. This work offers novel insights into the antioxidant mechanisms of resveratrol within biological systems and holds significant theoretical and practical implications for the medical application and development of resveratrol.

Sensitive detection of pymetrozine residues based on β-lactoglobulin as a fluorescence probe

A method was developed for the detection of pymetrozine (PYM) using β-lactoglobulin (β-LG) as a probe. The interaction between PYM and β-LG was studied by fluorescence spectra, circular dichroism spectra (CD), synchronous fluorescence spectra, ultraviolet visible spectra (UV–vis), fourier transform infrared (FT-IR) and molecular docking. The fluorescence quenching type between PYM and β-LG was demonstrated to be static by Stern-Volmer analysis and time-resolved fluorescence. The binding constant (Ka) was 2.95 × 104 L mol−1 at 293 K. The electrostatic attraction was confirmed to be the primary binding force by Ross theory, and the binding distance was 2.02 nm by fluorescence Resonance Energy Transfer (FRET) theory. Based on the interaction between β-LG and PYM, a fluorescence detection method for PYM was established. Under optimal experimental conditions, the standard equations were established in the range of 0.43 – 10.68 μg mL−1. The limit of detection (LOD) for PYM was 0.03 μg mL−1. The method was successfully applied to apple and pear fruit samples. Recoveries of PYM in these samples were 97.44 % – 102.29 %, the relative standard deviation (RSD) of these samples were 0.24 % – 0.88 % (n = 3).

Exploring the molecular binding mechanism of 6-fluoro, 4-hydroxy, 2- methyl quinoline with TiO2 nanoparticles: A spectroscopic, thermodynamic, and insights into the solvatochromic effect.

This study investigates the interaction between titanium oxide nanoparticles (TiO2 NPs) and the heterocyclic fluorophore 6-fluoro,4-hydroxy,2-methylquinoline (6-FHMQ), aiming to understand fluorescence quenching mechanisms and thermodynamic characteristics. Spectroscopic techniques including spectrofluorometry (FL) and spectrophotometry (UV-Vis) were used, with a lifetime decay (τ) of 0.18ns for 6-FHMQ measured using time correlated single photon counting (TCSPC). The interaction between 6-FHMQ and TiO2 NPs revealed a mix of static and dynamic fluorescence quenching mechanisms, with increasing quenching constants (Ksv) and a higher bimolecular quenching rate constant (Kq). The dynamic nature was highlighted by a temperature-dependent increase in binding sites from 1 to ~ 2. Spontaneous complexation was affirmed by negative change in free energy (ΔG), with negative change in enthalpy (ΔH) and a positive change in entropy (ΔS) values indicating favorable electrostatic and ionic interactions. The impact of varying TiO2 NP concentrations on 6-FHMQ absorption was analyzed using the Benesi-Hildbrand equation, with a quantum yield of 0.61 determined. By forster resonance energy transfer (FRET) theory, the proximity between 6-FHMQ and TiO2 NPs was found to be less than 70Å. Ground and excited state dipole moments of 6-FHMQ in different solvents were calculated to demonstrate solvent sensing ability and charge transfer properties. Ultimately, this study serves as a testament to the power of scientific innovation in the realms of drug delivery and tissue engineering.

Resonance energy transfer in orthogonally arranged chromophores: a question of molecular representation.

Energy transfer between orthogonally arranged chromophores is typically considered impossible according to conventional Förster resonance energy transfer theory. Nevertheless, the disruption of orthogonality by nuclear vibrations can enable energy transfer, what has prompted the necessity for formal expansions of the standard theory. Here, we propose that there is no need to extend conventional Förster theory in such cases. Instead, a more accurate representation of the chromophores is required. Through calculations of the energy transfer rate using structures from a thermal ensemble, rather than relying on equilibrium geometries, we show that the standard Förster resonance energy transfer theory is still capable of describing energy transfer in orthogonally arranged systems. Our calculations explain how thermal vibrations influence the electronic properties of the states involved in energy transfer, affecting the alignment of transition dipole moments and the intensity of transitions.

A composite electrodynamic mechanism to reconcile spatiotemporally resolved exciton transport in quantum dot superlattices.

Quantum dot (QD) solids are promising optoelectronic materials; further advancing their device functionality requires understanding their energy transport mechanisms. The commonly invoked near-field Förster resonance energy transfer (FRET) theory often underestimates the exciton hopping rate in QD solids, yet no consensus exists on the underlying cause. In response, we use time-resolved ultrafast stimulated emission depletion (STED) microscopy, an ultrafast transformation of STED to spatiotemporally resolve exciton diffusion in tellurium-doped cadmium selenide-core/cadmium sulfide-shell QD superlattices. We measure the concomitant time-resolved exciton energy decay due to excitons sampling a heterogeneous energetic landscape within the superlattice. The heterogeneity is quantified by single-particle emission spectroscopy. This powerful multimodal set of observables provides sufficient constraints on a kinetic Monte Carlo simulation of exciton transport to elucidate a composite transport mechanism that includes both near-field FRET and previously neglected far-field emission/reabsorption contributions. Uncovering this mechanism offers a much-needed unified framework in which to characterize transport in QD solids and additional principles for device design.

Elucidating the significance of molecular interaction between sulphur doped zinc oxide nanoparticles and serum albumin using multispectroscopic approach.

Ingenious nanomaterials with improved biocompatibility and multifunctional properties are gaining vital significance in biomedical applications, including advanced drug delivery and nanotheranostics. In a biological system, these nanoparticles interact with serum proteins forming a dynamic corona that affects their biological or toxicological properties producing undesirable effects. Thus, the current study focuses on the synthesis of sulphur-doped zinc oxide nanoparticles (ZnO/S NPs) and characterizing their mechanism of interaction with serum proteins using multispectroscopic approach. ZnO/S NPs were synthesized by employing a co-precipitation approach and characterized using various analytical techniques. The results of interaction studies demonstrated that ZnO/S NPs interact with serum albumins via the static quenching process. Analysis of thermodynamic parameters (ΔG, ΔH and ΔS) revealed that the binding process is spontaneous, exothermic and van der Waals force or hydrogen bonding plays a major role. The interaction of ZnO/S NPs with tyrosine residue in bovine serum albumin was established by synchronous fluorescence spectroscopy. In addition, the results of UV-visible, circular dichroism, Fourier transform infrared, Forster's resonance energy transfer theory and dynamic light scattering spectroscopic studies revealed that the ZnO/S NPs interact with albumin by inducing the conformational changes in secondary structure and reducing the α-helix content.

Waveguided energy transfer in pseudo-two-dimensional systems.

Resonance energy transfer (RET) is an important and ubiquitous process whereby energy is transferred from a donor chromophore to an acceptor chromophore without contact via Coulombic coupling. There have been a number of recent advances exploiting the quantum electrodynamics (QED) framework for RET. Here, we extend the QED RET theory to investigate whether real photon exchange can allow for excitation transfer over very long distances if the exchanged photon is waveguided. To study this problem, we consider RET in two spatial dimensions. We derive the RET matrix element using QED in two dimensions, consider an even greater confinement by deriving the RET matrix element for a two-dimensional waveguide using ray theory, and compare the resulting RET elements in 3D and 2D and for the 2D waveguide. We see greatly enhanced RET rates over long distances for both the 2D and 2D waveguide systems and see a great preference for transverse photon mediated transfer in the 2D waveguide system.

A Comparative Study of Nanobio Interaction of Zn-Doped CdTe Quantum Dots with Lactoferrin Using Different Spectroscopic Methods.

In this paper, glutathione (GSH)-coated Zn-doped CdTe quantum dots (QDs) with different particle sizes were synthesized using the "reflow method", and the interaction mechanism between the two QDs and lactoferrin (LF) was investigated systemically with different spectroscopic methods. The steady-state fluorescence spectra showed that the LF formed a tight complex with the two QDs through static bursting and that the electrostatic force was the main driving force between the two LF-QDs systems. The complex generation process was found to be spontaneous (ΔG < 0) and accompanied by exothermic and increasing degrees of freedom (ΔH < 0, ΔS > 0) by using the temperature-dependent fluorescence spectroscopy. The critical transfer distance (R0) and donor-acceptor distance (r) of the two LF-QDs systems were obtained based on the fluorescence resonance energy transfer theory. In addition, it was observed that the QDs changed the secondary and tertiary structures of LF, leading to an increase in the hydrophobicity of LF. Further, the nano-effect of orange QDs on LF is much larger than that of green QDs. The above results provide a basis for metal-doped QDs with LF in safe nano-bio applications.

Polariton-assisted resonance energy transfer beyond resonant dipole-dipole interaction: A transition-current-density approach

Using electric dipoles to describe light-matter interactions between two entities is a conventional approximation in physics, chemistry, and material sciences. However, the lack of material structures makes the approximation inadequate when the size of an entity is comparable to the spatial extent of electromagnetic fields or the distance between two entities. In this study, we develop a unified theory of radiative and non-radiative resonance energy transfer based on transition current density in a theoretical framework of macroscopic quantum electrodynamics. The proposed theory allows us to describe polariton-assisted resonance energy transfer between two entities with arbitrary material structures in spatially dependent vacuum electric fields. To demonstrate the generality of the proposed theory, we rigorously prove that our theory can cover the main results of the transition density cube method and the plasmon-coupled resonance energy transfer. We believe that this study opens a promising direction for exploring light-matter interactions beyond the scope of electric dipoles and provides new insights into material physics.

Macroscopic quantum electrodynamics theory of resonance energy transfer involving chiral molecules

Resonance energy transfer between chiral molecules can be used to discriminate between different enantiomers. The transfer rate between chiral molecules consists of a non-discriminatory and discriminatory parts. We derive these two rate contributions in the framework of macroscopic quantum electrodynamics. We show that their ratio is usually larger in the retarded regime or far-zone of large separation distances and that the degree of discrimination can be modified when considering a surrounding medium. We highlight the importance of local field effects onto the degree of discrimination and predict for general identical chiral molecules the optimum dielectric medium for discrimination. We apply our results on to 3-methylcyclopentanone and show that exotic media can even invert the discriminatory effect.

An efficient time-domain implementation of the multichromophoric Förster resonant energy transfer method.

The excitation energy transfer (EET) process for photosynthetic antenna complexes consisting of subunits, each comprised of multiple chromophores, remains challenging to describe. The multichromophoric Förster resonance energy transfer theory is a popular method to describe the EET process in such systems. This paper presents a new time-domain method for calculating energy transfer based on the combination of multichromophoric Förster resonance energy transfer theory and the Numerical Integration of the Schrödinger Equation method. After validating the method on simple model systems, we apply it to the Light-Harvesting antenna 2 (LH2) complex, a light harvesting antenna found in purple bacteria. We use a simple model combining the overdamped Brownian oscillators to describe the dynamic disorder originating from the environmental fluctuations and the transition charge from the electrostatic potential coupling model to determine the interactions between chromophores. We demonstrate that with this model, both the calculated spectra and the EET rates between the two rings within the LH2 complex agree well with experimental results. We further find that the transfer between the strongly coupled rings of neighboring LH2 complexes can also be well described with our method. We conclude that our new method accurately describes the EET rate for biologically relevant multichromophoric systems, which are similar to the LH2 complex. Computationally, the new method is very tractable, especially for slow processes. We foresee that the method can be applied to efficiently calculate transfer in artificial systems as well and may pave the way for calculating multidimensional spectra of extensive multichromophoric systems in the future.

A Comprehensive Approach to Exciton Delocalization and Energy Transfer.

Electrostatic intermolecular interactions lie at the heart of both the Förster model for resonance energy transfer (RET) and the exciton model for energy delocalization. In the Förster theory of RET, the excitation energy incoherently flows from the energy donor to a weakly coupled energy acceptor. The exciton model describes instead the energy delocalization in aggregates of identical (or nearly so) molecules. Here, we introduce a model that brings together molecular aggregates and RET. We will consider a couple of molecules, each described in terms of two diabatic electronic states, coupled to an effective molecular vibration. Electrostatic intermolecular interactions drive energy fluxes between the molecules, that, depending on model parameters, can be described as RET or energy delocalization. At variance with the standard Förster model for RET and of the exciton model for aggregates, our approach applies both in the weak and in the strong coupling regimes and fully accounts for the quantum nature of molecular vibrations in a nonadiabatic approach. Coupling the system to a thermal bath, we follow RET and energy delocalization in real time and simulate time-resolved emission spectra.

Spectroscopic and molecular docking studies of the interactions of sunset yellow and allura red with human serum albumin

AbstractBinding interactions of human serum albumin (HSA) with sunset yellow (SY) and allura red (AR), two food colorants, were investigated at the molecular level through fluorescence and UV absorption as well as molecular docking. The collective results of the study under the simulated physiological conditions proposed a static type of binding occurring between the two dyes and HSA. When compared with AR (293 K: Ksv = (4.21 ± 0.36) × 104 L·mol−1; Kb = (0.30 ± 0.23) × 106 L·mol−1), SY (293 K: Ksv = (6.80 ± 0.10) × 104 L·mol−1; Kb = (3.11 ± 2.01) × 106 L·mol−1) had stronger quenching ability and higher affinity for HSA due to less steric hindrance. It can be deduced that the energy transfer from HSA to the two dyes occurred with high probability based on the Förster resonance energy transfer theory (r &lt; 7 nm, 0.5 R0 &lt; r &lt; 2.0 R0). The spectral analysis suggested that the formation of the dye‐HSA complex resulted in the change in microenvironment around Tyr and Trp residues and in the secondary structure of the protein. According to molecular docking simulation, the two structural analogs almost bound to the same site of HSA, near Sudlow's Site I, but significant difference existed in the number and location of hydrogen bond (H‐bond) formed between the dyes and HSA. From the molecular docking along with the thermodynamic parameters (AR: ΔHo = −(58.79 ± 15.24) kJ·mol−1, ΔSo = −(115.1 ± 31.10) J·mol−1·K−1; SY: ΔHo = −(52.24 ± 3.15) kJ·mol−1, ΔSo = −(50.07 ± 11.14) J·mol−1·K−1), it could be inferred that H‐bond and van der Waals forces were the major binding forces involved in formation of the dye‐HSA complexes.

Generalized Resonance Energy Transfer Theory: Applications to Vibrational Energy Flow in Optical Cavities.

A general rate theory for resonance energy transfer (gRET) is formulated to incorporate any degrees of freedom (e.g., rotation, vibration, exciton, and polariton) as well as coherently coupled composite donor or acceptor states. The compact rate expression allows us to establish useful relationships: (i) detailed balance condition when the donor and acceptor are at the same temperature; (ii) proportionality to the product of dipole correlation tensors, which is not necessarily equivalent to spectral overlap; (iii) scaling with the effective coherent size, i.e., the number of coherently coupled molecules or modes; (iv) decomposition of collective rate in homogeneous systems into the monomer and coherence contributions such that the ratio of the two defines the quantum enhancement factor F; (v) spatial and orientational dependences as derived from the interaction potential. For the special case of exciton transfer, the general rate formalism reduces to FRET or its multichromophoric extension. When applied to cavity-assisted vibrational energy transfer between molecules or within a molecule, the general rate expression provides an intuitive explanation of intriguing phenomena such as cooperativity, resonance, and nonlinearity in the collective vibrational strong coupling (VSC) regime, as demonstrated in recent simulations. The relevance of gRET to cavity-catalyzed reactions and intramolecular vibrational redistribution is discussed and will lead to further theoretical developments.

Exploring the HSA/DNA/lung cancer cells binding behavior of p-Synephrine, a naturally occurring phenyl ethanol amine with anti-adipogenic activity: multi spectroscopic, molecular dynamic and cellular approaches

As a phenolic amine, p-Synephrine (SN) is known to exhibit lipolytic properties as well as enhancing energy expenditure, which can be an excellent alternative to ephedra as a dietary supplement. This study aimed to explore the subtle yet profound complex qualities and aspects of the interaction between SN, human serum albumin (HSA), and calf thymus DNA (ctDNA). To achieve a full comprehension on these fine-point distinctions, a nuanced and detail-oriented approach was implemented through a combination of different biophysical methods and molecular dynamics simulation techniques. The fluorescence spectroscopy revealed the binding of SN to HSA that led to the inducement of conformational changes. The quenching constants (KSV) of SN-HSA complex at 298, 303, and 308 K and were evaluated to be 2.95 × 104, 2.21 × 104 and 0.85 × 104 M−1, respectively. The thermodynamic parameters indicated the spontaneous formation of SN-HSA complex along with its regulation through H-bonding and van der Waals forces. The distance between SN and Trp residue of HSA was estimated at 3.91 nm by the Forster’s theory of non-radiative resonance energy transfer. According to CD spectroscopy, the binding of SN to HSA results in stabilization and promotes its functionality. Various spectroscopy methods and viscosity measurements proved the propensity of SN for binding to the grooves of ctDNA. Thermodynamic parameters indicate that the SN binds to DNA via a spontaneous and entropy-driven process that is mainly regulated by hydrophobic interactions. In conformity to the molecular modeling of HSA-SN complex, SN can be located in the active site of HSA and make a firm interaction, while the outcomes of ctDNA-SN interaction also indicated its tendency towards binding in DNA groove. In comparison to normal cells, SN caused a more pronounced reduction in lung cancer cell viability by increasing the expression of Bax and p53 at the mRNA and protein levels. Moreover, the incubation of H460 cells with the IC50 concentration of SN induced a notable decease in mRNA levels of PI3K, AKT, and mTOR, as well as protein levels of PI3Kγ-P110, P-AKT, and P-m TOR. These data indicate the potential of SN in binding with HSA, which reduces the proliferation of lung cancer cells mediated by apoptosis through the down regulation of PI3K/AKT/m TOR signaling pathway.

Enhanced photoluminescence of active ions in rugate type multilayer structures

Multilayer rugate-like structures based on porous silicon with incorporated active ions are theoretically investigated. Multilayer structures consist on different sinusoidal refractive index profiles designed to show the greatest amplification of electric field at a wavelength of 1 µm. We show that it is possible to increase the localization of the electric field by optimizing the design parameters, and thereby directly enhances the photoluminescence of the active ions. The luminescent properties of active ions incorporated in the porous silicon structures are calculated from a model based on the Förster Resonance Energy Transfer (FRET) theory. This model considers the effect of the electric field due to the structures and the energy transfer processes between donors and acceptors and allows studying the luminescent dynamics in this type of systems. It is shown that a greater intensity in the emission of ions is obtained from the structures with the highest amplification of the electric field. Furthermore, it is observed that the emission of active ions depends on the porous silicon multilayer structure and on the concentration of ions. Likewise, greater luminescence is observed as the acceptor density increases. In the same way, donor and acceptor lifetimes also depend on the acceptor density, although it does not depend on the porous silicon structure. The structures proposed here are prospective for Si-based optoelectronic applications for the near-infrared spectral region.

Binding of bioactive esculin and esculetin with hen egg white lysozyme: Spectroscopic and computational methods to comprehensively elucidate the binding affinities, interacting forces, and conformational alterations at molecular level

The binding of bioactive esculin and esculetin with lysozyme have been studied using multi-spectroscopic and computational methods. Intrinsic fluorescence of lysozyme was quenched via static quenching mechanism by these ligands and binding was observed to be moderate in nature. Structural alteration of the protein was noted upon ligand binding and enzymatic activity of lysozyme was also influenced in vitro. • Coumarin derivatives, esculin and esculetin quenched intrinsic fluorescence of HEWL through a static quenching mechanism. • Trp residue occupied a more hydrophobic environment upon interactions with esculin and esculetin. • The α-helical content of HEWL decreased in presence of ligands which could have affected the enzymatic activity of HEWL. • Δ G ° values were observed to be negative which indicated complex formation occurred spontaneously. Multi-spectroscopic and computational methods were employed to investigate the binding mechanism of esculin and esculetin with hen egg white lysozyme (HEWL). UV–vis and fluorescence spectroscopy proved static quenching mechanism was involved in the fluorescence quenching process. The binding constant ( K b ) determined for HEWL-esculin/esculetin complex was in the order of 10 4 M −1 . A negative Δ H ° [-(14.19 ± 2.56) kJ mol −1 ] and positive Δ S ° [+(40.98 ± 3.10) J K −1 mol −1 ] for HEWL-esculin complex suggested the presence of hydrophobic forces and hydrogen bonding. Whereas positive values of both Δ H° [+(9.96 ± 4.26) kJ mol −1 ] and Δ S ° [+(120.05 ± 14.14) J K -1 mol −1 ] for HEWL-esculetin complex indicated the presence of hydrophobic forces in the binding process. Blue shifts were observed for HEWL-esculin/esculetin complexes on carrying out synchronous and three-dimensional (3-D) fluorescence. A red edge excitation shift (REES) of 3 nm was observed for HEWL, whereas 6 and 5 nm for HEWL-esculin and HEWL-esculetin complexes, respectively, were also measured. Circular dichroism (CD) studies revealed that the % α-helical content of HEWL (25.56 ± 2.24) decreased in the presence of esculin (17.96 ± 1.54) and esculetin (19.90 ± 2.14). Amide I peak of HEWL, determined from Fourier transform infrared (FTIR) studies, shifted from 1657 cm −1 to 1654 cm −1 and 1652 cm −1 upon complexation with esculin/esculetin, respectively. The binding distance ( r ) for HEWL-esculin (4.40 ± 0.14 nm) and HEWL-esculetin (4.66 ± 0.11 nm) complexes were determined from fluorescence resonance energy transfer (FRET) theory. Thermal analysis resulted in a lower T m value for the protein–ligand complexes (71.8 °C) compared to the native protein (73.3 °C). Molecular docking and molecular dynamic (MD) simulations provided insight into the binding affinity and flexibility of HEWL upon complexation, respectively. In the presence of esculin and esculetin, a change in enzyme activity of HEWL was observed.

Evaluation of interaction between Ponceau 4R (P4R) and trypsin using kinetic, spectroscopic, and molecular dynamics simulation methods

Azo-dyes, a diverse group of synthetic dyes, are commonly used for coloring food products. However, these dyes are persistent in nature with the potential to cause harmful effects on the human body. Hence, it seems that studies are required about the effects of azo dyes on the structure and function of biomacromolecules. Ponceau 4R (P4R) is an azo-dye found in foods, beverages, and pharmaceuticals, but its potential risk on gastrointestinal enzymes such as trypsin is poorly characterized. Thus, this study focused on analyzing the binding behavior between trypsin and P4R by spectroscopic, kinetic, and theoretical methods. Intrinsic fluorescence revealed that there was an interaction between P4R and trypsin, and P4R could quench the trypsin fluorescence by a static model. The resonance light scattering (RLS) method was used to confirm the formation of the P4R-trypsin complex. The thermodynamic data indicated that the predominant interactions between P4R and trypsin were Van der Waals and hydrogen bonds, consistent with docking results. The distance between P4R and trypsin after the interaction was calculated based on fluorescence resonance energy transfer (FRET) theory and found to be 3.36 nm. Synchronous fluorescence, three-dimensional fluorescence, UV–Visible spectrophotometry, and circular dichroism (CD) analyses indicated that the conformation of trypsin was changed upon binding with P4R, which could affect the trypsin function. Kinetic studies revealed that P4R inhibited trypsin activity in a non-competitive model, and docking analysis confirmed that P4R was not bound to the active site of the trypsin. The molecular dynamics (MD) simulation studies showed that the root mean square deviation (RMSD) in the trypsin-P4R complex (0.270 ± 0.02 nm) was larger than that of the free trypsin system (0.221 ± 0.01 nm), which agreed with CD and thermal stability experiments. Thus, P4R was shown to be a potent inhibitor and quencher of trypsin.