Fluorescence Spectroscopic Studies Research Articles

Bioactive porphyrin magnesium (II) complex. Synthesis, molecular structure and spectroscopic characterization. Biological activity and molecular docking studies

We have synthesized and characterized porphyrin structures by UV–visible, IR and fluorescence spectroscopy studies and single crystal XRD analysis; H2(TPP) (I) (meso-tetraphenylporphyrin), [Mg(TPP)(DMF)] (II); (DMF: dimethylformamide) and [Mg(TPP)(H2O)]0.2(4-CNpy) (III); (4-CNpy: 4-Cyanopyridine). The crystal packing of the porphyrin structures is stabilized by intermolecular hydrogen bonds and by intermolecular CH⋯Cg π interactions where Cg is the centroid of the phenyl and pyrrole rings. UV–Vis spectroscopic data confirmed the creation of Mg-meso-porphyrin complexes by the red-shifted Soret bands for our derivatives compared to the free-base porphyrin. The optical gap energy values of (I), (II) and (III) are 1.92, 2.07 and 2.00 eV. Gram-positive and Gram-negative bacteria were tested against free porphyrin (I), metallated porphyrin (II) and complex (III). The results show that the magnesium porphyrin derivative is highly effective compared to H2TPP free-base porphyrins and metallated porphyrin [Mg(TPP)] and has higher inhibition character against the tested bacteria. The molecular docking of the complex (III), in the active sites of bacterial proteins was carried out to detect the degree of antibacterial activity of recognition.

Thermodynamics of the Eu(III)-Mg-SO4-H2O and Eu(III)-Na-SO4-H2O systems. Part II: spectroscopy experiments, complexation and Pitzer/SIT models.

A time-resolved laser fluorescence spectroscopy (TRLFS) study was carried out to investigate the Eu(III)-SO4 complexation at room temperature over a wide range of Na2SO4 concentrations (0-2 mol kg-1). Spectroscopic observations confirm the step-wise formation of the aqueous complexes Eu(SO4)+, Eu(SO4)2- and Eu(SO4)33- over the investigated Na2SO4 concentrations. Combining TRLFS data obtained in this study and solubility data reported in Part I of this work for the Eu2(SO4)3-Na2SO4-H2O and Eu2(SO4)3-MgSO4-H2O systems, thermodynamic and activity models were derived based on the SIT and Pitzer formalisms. A combination of the geochemical calculation codes PhreeqC (SIT), PhreeSCALE (Pitzer) and the parameter estimation code PEST was used to determine the solubility products of Eu2(SO4)3·8H2O(cr) and Na2Eu2(SO4)4·2H2O(cr), stability constants of the Eu(III)-SO4 complexes (β0i), and the specific binary and ternary interaction parameters (εij, β(0)ij, β(1)ij, Cϕij, θik, Ψijk) for both activity models. The thermodynamic constants determined in this work are discussed with reference to values available in the literature.

Dendrimer Induced Bilayer Disintegration of Hybrid Vesicles.

Physicochemical investigations on the inclusion of anionic polyamidoaminesuccinamic acid dendrimer, generation 5 (PAMAM-SA, G5) with positively charged hybrid vesicles (HCV), prepared using soylecithin, ion pair amphiphile (IPA), cholesterol and dihexadecyldimethylammonium bromide, were investigated by dynamic light scattering, transmission electron/atomic force microscopy (TEM/AFM), differential scanning calorimetry, fluorescence spectroscopy and surface pressure-time isotherm studies. Adsorption of dendrimer onto vesicle surface and subsequent bilayer disruption strongly depends on the bilayer composition and dendrimer concentration. Change in the zeta potential value with increasing dendrimer concentration suggests the dendrimer-vesicle interaction to be electrostatic in nature. AFM studies also confirm the adsorption of dendrimer as well as hole formation in the bilayer. Impact of the inclusion of dendrimer into the bilayer were further investigated through differential scanning calorimetry by monitoring the chain melting temperature and enthalpy of the chain melting processes. Dendrimer at low concentration does not alter bilayer integrity, while hole formations are noted at higher dendrimer concentration. Fluorescence anisotropy studies confirm the adsorption and subsequent bilayer disruption due to dendrimer inclusion. Dendrimer induced vesicle disintegration kinetics conclusively illustrate the transformation of cationic bilayer to monolayer and thereby exposing the role of IPA. In vitro cytotoxicity studies on PAMAM-SA, G5 and HCVs mixtures against human breast cancer cell line suggest that dendrimer-liposome aggregates (dendriosomes) exhibit substantial anticancer activities with insignificant side effects. It is expected that the dendriosomes may have application to host and deliver anticancer drug in the field of targeted drug delivery.

Comparison of In Vivo Study of Co-Polarized and Cross-Polarized Fluorescence Spectroscopy to Co-Minus Cross-Polarized Fluorescence Spectroscopy to Diagnose Precancerous Cervical Lesions by Handheld Probe

Comparison of In Vivo Study of Co-Polarized and Cross-Polarized Fluorescence Spectroscopy to Co-Minus Cross-Polarized Fluorescence Spectroscopy to Diagnose Precancerous Cervical Lesions by Handheld Probe

Rotaxane-Functionalized Dyes for Charge-Rectification in p-Type Photoelectrochemical Devices.

A supramolecular photovoltaic strategy is applied to enhance power conversion efficiencies (PCE) of photoelectrochemical devices by suppressing electron-hole recombination after photoinduced electron transfer (PET). Here, the author exploit supramolecular localization of the redox mediator-in close proximity to the dye-through a rotaxane topology, reducing electron-hole recombination in p-type dye-sensitized solar cells (p-DSSCs). Dye PRotaxane features 1,5-dioxynaphthalene recognition sites (DNP-arms) with a mechanically-interlocked macrocyclic redox mediator naphthalene diimide macrocycle (3-NDI-ring), stoppering synthetically via click chemistry. The control molecule PStopper has stoppered DNP-arms, preventing rotaxane formation with the 3-NDI-ring. Transient absorption and time-resolved fluorescence spectroscopy studies show ultrafast (211 ± 7 fs and 2.92 ± 0.05ps) PET from the dye-moiety of PRotaxane to its mechanically interlocked 3-NDI-ring-acceptor, slowing down the electron-hole recombination on NiO surfaces compared to the analogue . p-DSSCs employing PRotaxane (PCE = 0.07%) demonstrate a 30% PCE increase compared to PStopper (PCE = 0.05%) devices, combining enhancements in both open-circuit voltages (VOC = 0.43 vs 0.36V) and short-circuit photocurrent density (JSC = -0.39vs -0.34mA cm-2 ). Electrochemical impedance spectroscopy shows that PRotaxane devices exhibit hole lifetimes (τh ) approaching 1 s, a 16-fold improvement compared to traditional I- /I3 - -based systems (τh = 50ms), demonstrating the benefits obtained upon nanoengineering of interfacial dye-regeneration at the photocathode.

A comparative study of fluorescence and Raman spectroscopy for discrimination of virgin olive oil categories: Chemometric approaches and evaluation against other techniques

Spectroscopic techniques are presented as a reliable and economical alternative for the discrimination of olive oil quality categories. In this work, fluorescence and Raman spectroscopic techniques have been compared by evaluating different characteristics focused on sample preparation and analysis time, as well as classification percentages by chemometric models (PLS-DA) to classify olive oils into three categories: extra virgin (EVOO), virgin (VOO), and lampante (LOO). A statistical evaluation of the quality of the chemometric models was performed by comparing the parameters of sensitivity in terms of the true positive rate (%TPR), specificity (or true negative rate %TNR), and classification error in calibration, cross-validation and prediction. Fluorescence spectroscopy was the screening technique with the best overall accuracy in the ternary models for prediction (78.60%), with higher results than those obtained by Raman (66.03%), the latter being below the average error in the tasting panels. Regarding the classification obtained in binary models (EVOO/Non-EVOO and LOO/Non-LOO), fluorescence spectroscopy continued to present better results than Raman, with classifications of 92.5% and 88.5 % for both models, respectively (Raman obtained percentages of 70.7% and 66.6%). A comparison of the results obtained with confirmation techniques (headspace-gas chromatography-ion mobility spectrometry, HS-GC-IMS) and other spectroscopic techniques (NIR and MIR) studied in previous research was carried out, where it was observed that fluorescence spectroscopy obtained the closest classification percentage to the HS-GC-IMS technique (86.4%) in ternary models. On the other hand, the NIR technique obtained the highest classification percentage in binary models, with 92.6% for EVOO/Non-EVOO and 88.9% for LOO/Non-LOO compared to the value obtained using GC-IMS (95.4 and 86.4 %). Evaluating the rest of the parameters of interest for the olive industry, IR (both NIR and MIR) was the most interesting technique because it is a cheap, simple sensor with fast chemometric treatments and is environmentally-friendly. The results of this comparative study can be used to provide solutions and alternatives for applying new analytical strategies to support the work of tasting panels.

Synthesis and Properties of Fluorenone-Containing Cycloparaphenylenes and Their Late-Stage Transformation.

Cycloparaphenylenes (CPPs) are the smallest possible armchair carbon nanotubes, the properties of which strongly depend on their ring size. They can be further tuned by either peripheral functionalization or by replacing phenylene rings for other aromatic units. Here we show how four novel donor-acceptor chromophores were obtained by incorporating fluorenone or 2-(9H-fluoren-9-ylidene)malononitrile into the loops of two differently sized CPPs. Synthetically, we managed to perform late-stage functionalization of the fluorenone-based rings by high-yielding Knoevenagel condensations. The structures were confirmed by X-ray crystallographic analyses, which revealed that replacing a phenylene for a fused-ring-system acceptor introduces additional strain. The donor-acceptor characters of the CPPs were supported by absorption and fluorescence spectroscopic studies, electrochemical studies (displaying the CPPs as multi-redox systems undergoing reversible or quasi-reversible redox events), as well as by computations. The oligophenylene parts were found to comprise the electron donor units of the macrocycles and the fluorenone parts the acceptor units.

Synthetic dimethoxyxanthones bind similarly to human serum albumin compared with highly oxygenated xanthones

Spectroscopic studies were carried out for the interaction of the bioactive compounds 2,3-dimethoxyxanthone (1) and 3,4-dimethoxyxanthone (2) with human serum albumin (HSA). The UV absorption, steady-state, and time-resolved fluorescence spectroscopy studies showed that 1 and 2 interact with HSA through a ground-state association. The decrease in the Stern-Volmer constant (KSV) values with increasing temperature, and the bimolecular quenching rate constant (kq) values in the order of 1012 M−1s−1 indicated a static fluorescence quenching mechanism, corroborating with time-resolved fluorescence decays. The association constant (Ka) values in the order of 104 M−1 indicated a moderate interaction between these xanthones and HSA. Circular dichroism experiments showed weak perturbation on the secondary structure of albumin after the interaction with 1 and 2. Thermodynamic parameters suggested that the binding is entropically (for HSA:1, ΔS° 0.012 ± 0.003 and for HSA:2 0.008 ± 0.002 J/molK) and enthalpically (for HSA:1, ΔH° -24.0 ± 0.99 and for HSA:2 -25.6 ± 0.67 kJ/mol) controlled, with a Gibbs free energy change (ΔG°) close to -28.0 kJ/mol in both cases. Competitive drug-displacement and molecular docking results showed that 1 and 2 could interact not only with subdomain IIA, where Trp-214 residue can be found, but also with subdomains IIIA and IB. Hydrogen bonding, electrostatic, and hydrophobic interactions were detected as the main binding forces to stabilize the association HSA:1 and HSA:2. Overall, the dimethoxyxanthones have similar binding affinity compared with highly oxygenated xanthones, e.g., mangiferin, gentiacaulein, and norswertianin.

Studies on anionic dye Eosin Y in mixed anionic micellar medium

Dye-Surfactant interactions in aqueous solution are important in industrial processes. Generally, dyes associate strongly with oppositely charged surfactants. But, similarly charged dye-surfactants are not preferred anticipating repulsion. To gain insight into the nature and extent of interaction between anionic dye Eosin Yellow (EY) and mixed anionic micellar system, this study was undertaken. Mixed micellar system comprised of anionic, Sodium dodecyl sulfate (SDS) and nonionic Pluronic L-35 (L-35). The characterizations employed were FTIR Spectroscopy, Conductivity, UV visible spectroscopy, Fluorescence spectroscopy and SEM studies. A red shift with enhancement of intensity was observed both in UV-absorption and emission spectra of EY when interacted with SDS + L-35 mixed micellar system. From the conductance measurements, the counter ion binding constant β was calculated. Subsequently, various thermodynamic parameters viz., ΔGm, ΔHm and ΔSm were evaluated using β. The Gibbs free energy of micellization, ΔGm was observed to be −4.56 kJ mol−1, −13.34 kJ mol−1 and −18.55 kJ mol−1 for SDS, SDS + L-35 and SDS + L-35 + EY respectively. The negative values of ΔGm are indicating the spontaneous binding occurring between EY and SDS + L-35. The SEM images revealed enlargement of grain size of the mixed micelle after encapsulation of EY. To the best of our knowledge this is the first report of anionic dye EY with an anionic mixed micellar system.

Albumin from sera of rheumatoid arthritis patients share multiple biochemical, biophysical and immunological properties with in vitro generated glyco-nitro-oxidized-albumin

The purpose of the present study is to explore the effects of endogenous stressors on structure and function of rheumatoid arthritis (RA) patients’ albumin. In contrast to glycated-albumin or nitro-oxidized-albumin, high titre antibodies against glyco-nitro-oxidized-albumin were found in the sera of RA patients. Also, compared to the other two modified forms of albumin, glyco-nitro-oxidized-albumin showed highest percent inhibition. Albumin isolated from RA patients’ sera displayed hyperchromicity and quenching of tyrosine and tryptophan fluorescence. Fluorescence spectroscopy studies also revealed the presence of dityrosine and advanced glycation end products in RA patient’s albumin. RA patients’ albumin showed weaker binding with 1-anilinonaphthalene-8-sulfonic acid dye. Secondary structure alterations were demonstrated by circular dichroism and Fourier transform infrared spectroscopy. Biochemical investigations revealed substantial decline in the availability of free side chains of amino acid residues; increased carbonyls and decreased sulfhydryls in RA patients’ albumin. The functional impairment in RA patients’ albumin was revealed by their low binding with bilirubin and cobalt. Liquid chromatography mass spectrometry analysis revealed the presence of Nε-(carboxymethyl) lysine and 3-nitrotyrosine in RA patients’ albumin. The amyloidogenic aggregation of RA patients’ albumin was confirmed by Congo red absorption and thioflavin-T fluorescence assays. The morphology of the aggregates was visualized under scanning and transmission electron microscope. From the above findings, we inferred that endogenous stress in RA patients have modified albumin and produce structural/functional abnormalities. Also, the presence of anti-glyco-nitro-oxidized-albumin antibodies along with other clinical features may be used as biomarker for the diagnosis and assessment of treatment responses in RA patients. Communicated by Ramaswamy H. Sarma

The Recent Report on Schiff Bases and their Complexes as DNA Binders

Abstract: Schiff bases are dynamic organic compounds of bioactive importance, acting as flexible ligands and can form complexes with metals. Schiff bases along with their metal complexes have shown a large affinity towards DNA, which is one of the main targets of chemotherapeutics. DNA can join hands with these motifs in multiple ways, like covalent (crosslinking adducts) and non-covalent (intercalative or groove binding via electrostatic) interactions. Besides, the binding ability and types vary with the metals involved in Schiff base metal complexes. Thus, this review aims to describe significant recent reports of Schiff bases and their metal complexes capable of binding with DNA to evaluate the superlative DNA binders, type of association, and best metal-Schiff base combinations. Among the experimental techniques employed in revealing these interactions, noteworthy UV absorption and fluorescence spectroscopy studies have been discussed for the assessment of various types of bindings as intercalation or groove type.

Rational surface charge engineering of haloalkane dehalogenase for boosting the enzymatic performance in organic solvent solutions

Biocatalysis in organic solvents (OSs) has numerous important applications, but native enzymes in OSs often exhibit limited catalytic performance. Herein, we proposed a computation-aided surface charge engineering strategy to improve the catalytic performance of haloalkane dehalogenase DhaA in OSs based on the energetic analysis of substrate binding to the DhaA surface. Several variants with enhanced OS resistance were obtained by replacing negative charged residues on the surface with positive charged residue (Arg). Particularly, a four-substitution variant E16R/E93R/E121R/E257R exhibited the best catalytic performance (five-fold improvement in OS resistance and seven-fold half-life increase in 40% (vol) dimethylsulfoxide). As a result, the overall catalytic performance of the variant could be at least 26 times higher than the wild-type DhaA. Fluorescence spectroscopy and molecular dynamics simulation studies revealed that the residue substitution mainly enhanced OS resistance from four aspects: (a) improved the overall structural stability, (b) increased the hydrophobicity of the local microenvironment around the catalytic triad, (c) enriched the hydrophobic substrate around the enzyme molecule, and (d) lowered the contact frequency between OS molecules and the catalytic triad. Our findings validate that computation-aided surface charge engineering is an effective and ingenious rational strategy for tailoring enzyme performance in OSs.

Assessing Partial Inhibition of Ribonuclease A Activity by Curcumin through Fluorescence Spectroscopy and Theoretical Studies.

Molecular interactions and controlled expression of enzymatic activities are fundamental to all cellular functions in an organism. The active polyphenol in turmeric known as curcumin (CCM) is known to exhibit diverse pharmacological activities. Ribonucleases(RNases) are the hydrolytic enzymes that plays important role in ribonucleic acid (RNA) metabolism. Uncontrolled and unwanted cleavage of RNA by RNases may be the cause of cell death leading to disease states. The protein ribonuclease A (RNase A) in the superfamily of RNases cleaves the RNA besides its role in different diseases like autoimmune diseases, and pancreatic disorders. Interaction of CCM with RNase A have been reported along with the possible role of CCM to inhibit the RNase A enzymatic activity. The interaction strength was found to be 104M-1 order from spectroscopic results. Quenching of RNase A fluorescence by CCM was 104M-1 order. Non-radiative energy transfer from RNase A (donor) to CCM (acceptor) suggested a distance of 2.42nm between the donor-acceptor pair. Circular dichroism studies revealed no structural changes in RNase A after binding. Binding-induced conformational variation in protein was observed from synchronous fluorescence studies. Agarose gel electrophoresis revealed a partial inhibition of the RNase A activity by CCM though not significant. Molecular docking and molecular dynamics studies suggested the residues of RNase A involved in the interaction with supporting the experimental finding for the partial inhibition of the enzyme activity. This study may help in designing new CCM analogues or related structures to understand their differential inhibition of the RNase A activity.

Dual-Stimuli-Activatable Hybrid Prodrug for the Self-Immolative Delivery of an Anticancer Agent and Hydrogen Sulfide with Turn-On Fluorescence.

Stimuli-responsive fluorogenic prodrugs are advantageous for the targeted drug delivery enabling real-time non-invasive monitoring with turn-on fluorescence. We report herein the dual-stimuli (ROS and CA)-responsive thiocarbamate-based prodrug (AM-TCB) for the turn-on fluorogenic delivery of the naphthalimide-based anticancer agent amonafide along with the gasotransmitter hydrogen sulfide (H2 S). A carbamate-based prodrug AM-CB was also designed, capable of releasing the anticancer agent amonafide without any H2 S. The prodrugs were synthesized using multi-step organic synthesis. UV-Vis and fluorescence spectroscopic studies revealed selective reactivity of the boronate ester group of prodrugs towards ROS (primarily H2 O2 ) with the release of amonafide and COS/CO2 via self-immolative processes. Hydrolysis of the generated COS by carbonic anhydrase (CA) produces H2 S. While the prodrug AM-TCB retained the anticancer activity of free amonafide in cancer cells (MDA-MB-231 and HeLa), unlike amonafide, it enhanced the cellular viability of the non-malignant cells (HEK-293). Fluorescence imaging in HeLa cells revealed the simultaneous delivery of the anticancer agent and H2 S from AM-TCB with turn-on fluorescence. Western blot studies further revealed the cytoprotective effects of the released H2 S from AM-TCB. The present adjuvant strategy therefore would be helpful in future for ameliorating the anticancer drug-induced side-effects.

Milk protein-based carrier system for encapsulation of dietary polyphenol rutin: Molecular-level interactions and enhanced bioactivities

The self-assembly process of proteins provides plenty of potential for redesigning and combining them into supramolecular assemblies for real-time applications. The present study demonstrates the encapsulation of dietary polyphenol rutin in β-casein (βCN) micellar cavity followed by their molecular interactions and biological implications. The prepared rutin-loaded βCN particles (DLS: 274.2 ± 3.86 nm; TEM: ∼200 nm) appeared to be smoothly spherical. Isothermal titration calorimetry (ITC) studies indicated that the encapsulation process was thermodynamically favorable and a typical example of entropy-driven hydrophobic interaction (ΔH: 38.1 kJ mol−1; TΔS = 51.9 kJ mol−1 K−1). The molecular interaction of rutin with βCN was characterized by absorption, steady-state fluorescence, and time-resolved fluorescence spectroscopic studies. The intrinsic fluorescence of βCN was quenched by rutin via a static mechanism with a favorable binding affinity. Molecular docking studies suggested the involvement of hydrophobic and hydrogen bonding interactions between the concerned entities. A sustained release mechanism followed by significant improvement in the cytotoxicity of rutin was observed after encapsulation. An in vitro digestion model mimicking gastric and intestinal phases was also employed to study the bioaccessibility. Our study may assist in formulating more efficient casein-based carrier systems to enhance the bio-efficacy of polyphenols.

Electrochemical Sensor Based on N–Doped Carbon Nanodots Derived from the Agro-Waste Cotton Boll Peel Extracts for Nano Molar Determination of an Anti-Cancer Drug, Pemetrexed

A green and facile one-step synthetic route was proposed for the synthesis of nitrogen-doped carbon nanodots (N-CDs) from agro-waste cotton boll peel extract as a natural carbon source and glycine as a nitrogen source. The prepared N-CDs were utilized further in the fabrication of an electrochemical sensor for the nanomolar determination of an anti-cancer drug, pemetrexed (PMT). The electrode material was characterized by XRD, FTIR, TEM, Raman, EDX, UV–vis, fluorescence, cyclic voltammetric and electrochemical impedance spectroscopic studies. The N-CDs drop-casted on the surface of glassy carbon electrode (GCE) served as a good sensing material and exhibited electrocatalytic activity as evident from ∼20-fold enhancement in the oxidation peak current of PMT at N-CDs/GCE with 130 mV lesser positive oxidation peak potential than that at bare GCE. This was attributed to excellent conductivity, higher electroactive surface area, graphitic cores of N-CDs besides the interactions between PMT and N-CDs via hydrogen bonding and π–π stacking. The sensing performance of the demonstrated electrode was optimized by varying the peak current dependant parameters and also by investigating the influence of interfering substances in the determination of PMT. The electrochemical behaviour of PMT at different pH revealed the adsorption controlled electrode process with the involvement of protons. Under optimized conditions, differential pulse voltammetric (DPV), square wave voltammetric (SWV) and adsorptive differential pulse voltammetric (AdSDPV) methods were developed for sensitive determination of PMT at nano molar level. Further, these developed analytical methods were applied successfully for the determination of PMT in analyte fortified human urine samples and pharmaceutical formulations.

Exploring the Structural Importance of the C3=C4 Double Bond in Plant Alkaloids Harmine and Harmaline on Their Binding Interactions with Hemoglobin.

Harmine and harmaline are two structurally similar heterocyclic β-carboline plant alkaloids with various therapeutic properties, having a slight structural difference in the C3=C4 double bond. In the present study, we have reported the nature of the interaction between hemoglobin (Hb) with harmine and harmaline by employing several multispectroscopic, calorimetric, and molecular docking approaches. Fluorescence spectroscopic studies have shown stronger interaction of harmine with Hb compared to that of almost structurally similar harmaline. Steady-state anisotropy experiments further show that the motional restriction of harmine in the presence of Hb is substantially higher than that of the harmaline-Hb complex. Circular dichroism (CD) study demonstrates no conformational change of Hb in the presence of both alkaloids, but CD study in 1-cm cuvette path length also demonstrates stronger affinity of harmine toward Hb compared to harmaline. From the thermal melting study, it has been found that both harmine and harmaline slightly affect the stability of Hb. From isothermal titration calorimetry (ITC), we have found that the binding process is exothermic and enthalpy driven. Molecular docking studies indicated that both harmine and harmaline prefer identical binding sites in Hb. This study helps us to understand that slight structural differences in harmine and harmaline can alter the interaction properties significantly, and this key information may help in the drug discovery processes.

Fluorescence Spectroscopic Studies to Evaluate Binding Interaction between Hoechst 33258 and Bilirubin.

A detailed spectroscopic study (fluorescence, absorption, and lifetime) was conducted to gain insight into the nature of the binding interaction between fluorophore Hoechst33258 (H258) and jaundice marker Bilirubin (Br). The fluorescence emission of the H258 (Ex/Em = 340-502nm) showed a conc. dependent quenching in the presence of Br (1.25[Formula: see text]M to 10[Formula: see text]M). The Stern-Volmer constant demonstrated an upward curve depicting the occurrence of both static and dynamic quenching with an acquired value of K[Formula: see text] = 3.1x 10[Formula: see text] M[Formula: see text] and biomolecular quenching rate constant K[Formula: see text] = 8.6 x 10[Formula: see text] M[Formula: see text]S[Formula: see text]. The static quenching was evaluated using the sphere of action model and a sphere radius of 0.3nm indicated the presence of a static component in the quenching. The FRET analysis with overlap integral (J) = 1.4x10[Formula: see text] M[Formula: see text]cm[Formula: see text]nm[Formula: see text] and Foster Radius(R[Formula: see text]) = 26.82 Å with 59% efficiency suggested occurrence of dynamic quenching. Further studies with the time-resolved fluorescence also indicated the presence of dynamic quenching. The lifetime values of H258 reduced from 3.9ns to 0.5ns. Molecular docking studies further support both static and dynamic components in quenching. A non-covalent interaction of H258 with Br in the presence of HSA is predominantly characterized by H-bonding with residues Lys, Asn, Glu, Gln, and Br. The H258 and Br interaction was within the distance of 3.04 Å, which is in coherence with the sphere of action model (0.3nm) and Van-der-Waals along with hydrophobic interactions, which suggested both static and dynamic quenching. Thus, H258 can serve as an efficient fluorophore to monitor binding interactions and can be further exploited as a suitable probe for investigating conformational changes and detection of Br in subsequent studies.

Importance of aspartate 4 in the Mg2+ dependent regulation of Leishmania major PAS domain-containing phosphoglycerate kinase

Magnesium is an important divalent cation for the regulation of catalytic activity. Recently, we have described that the Mg2+ binding through the PAS domain inhibits the phosphoglycerate kinase (PGK) activity in PAS domain-containing PGK from Leishmania major (LmPAS-PGK) at neutral pH 7.5, but PGK activity is derepressed at acidic pH 5.5. The acidic residue within the PAS domain of LmPAS-PGK is expected to bind the cofactor Mg2+ ion at neutral pH, but which specific acidic residue(s) is/are responsible for the Mg2+ binding is still unknown. To identify the residues, we exploited mutational studies of all acidic (twelve Asp/Glu) residues in the PAS domain for plausible Mg2+ binding. Mg2+ ion-dependent repression at pH 7.5 is withdrawn by substitution of Asp-4 with Ala, whereas other acidic residue mutants (D16A, D22A, D24A, D29A, D43A, D44A, D60A, D63A, D77A, D87A, and E107A) showed similar features compared to the wild-type protein. Fluorescence spectroscopic studies and isothermal titration calorimetry analysis showed that the Asp-4 is crucial for Mg2+ binding in the absence of both PGK's substrates. These results suggest that Asp-4 residue in the regulatory (PAS) domain of wild type enzymes is required for Mg2+ dependent repressed state of the catalytic PGK domain at neutral pH.

Experimental and computational investigation of the binding mechanism of thymol with human transferrin: Importance of dietary phytochemicals in Alzheimer’s disease therapeutics

Transferrin (Tf) regulates free iron in the body, which is critical to shield the body from the detrimental effects of free iron, a potent neurotoxin, in the aerobic surroundings of the blood and body fluids. Maintaining iron homeostasis is essential because any disruption in its homeostasis ultimately results in the generation of iron-triggered oxidative stress that is further implicated in the pathogenesis of numerous diseases, including Alzheimer’s disease (AD). In recent times, plant extracts and plant-derived phytochemicals have gained importance in the field of drug design and discovery. The present study aimed to comprehensively explore the interaction of thymol with Tf, owing to the pivotal roles played by Tf and thymol in AD. Initially, the details of the binding of thymol with Tf were assessed employing docking and all-atom molecular dynamics (MD) simulation studies for 300 ns in detail. It was found that thymol binds to Tf with a good affinity, forming close interactions with key residues, specifically within Tf's hydrogen carbonate and iron-binding domain. Moreover, the binding of thymol did not cause any significant structural changes in the Tf conformation, implying the Tf-thymol complex stability. The results of UV visible and fluorescence spectroscopic studies supported strong binding of thymol with Tf, signifying the stability of the Tf-thymol complex. The study provided valuable insights into the binding mechanisms, structural stability, and conformational dynamics of the Tf-thymol complex, providing a platform to use thymol and its derivatives in AD therapeutics.