Tryptophan Emission Research Articles

Rapid detection of bactericidal efficacy of nanoparticles coated polyurethane foam by synchronous fluorescence spectroscopy

The ability of right-angled synchronous fluorescence spectroscopy (SFS) was explored to analyse the bacterial load in water treated with green synthesized silver nanoparticles (AgNPs) coated polyurethane foam (PUF). Gram negative (Escherichia coli, Pseudomonas aeruginosa) and Gram positive (Staphylococcus aureus) bacteria cultured in nutrient broth were diluted in autoclaved water containing NPs-coated PUF. The survival rate of S. aureus and E. coli lowered after ten minutes as compared to P. aeruginosa; however, after thirty minutes, the percentage viability dropped and recorded as 3.4%, 0.9%, and 0.1% for E. coli, P. aeruginosa and S. aureus respectively in the treated suspensions. No spectral change was observed in the fluorescence emission from the positive control and treated bacterial suspension owing to the masking effect of the emission from nutrient broth. In parallel, SF spectra recorded for directly picked bacterial colony dissolved in water showed remarkable drop in tryptophan emission after treatment with NPs-coated PUF. The SF data changes were assisted by hierarchical cluster analysis, which also made it possible to distinguish between positive control and treated bacterial suspensions. SFS has shown to be a reliable substitute for the culture plate approach for the quick identification of bacterial contamination in water.

Addressing the interaction of stem bromelain with different anionic surfactants, below, at and above the critical micelle concentration (cmc) in phosphate buffer at pH 7: Physicochemical, spectroscopic, & molecular docking study

The structural stability and therapeutic activity of Stem Bromelain (BM) have been explored by unravelling the interaction of stem BM in presence of two different types of anionic surfactants namely, bile salts, NaC and NaDC and the conventional anionic surfactants, SDDS and SDBS, below, at and above the critical micelle concentration (cmc) in aqueous phosphate buffer of pH 7. Different physicochemical parameters like, surface excess (Γcmc), minimum area of surfactants at air water interface (Amin) etc. are calculated from tensiometry both in absence and presence of BM. Several inflection points (C1, C2 and C3) have been found in tensiometry profile of surfactants in presence of BM due to the conformational change of BM assisted by surfactants. Similar observation also found in isothermal titration calorimetry (ITC) profiles where the enthalpy of micellization (ΔH0obs) of surfactants in absence and presence of BM have calculated. Further, steady state absorption and fluorescence spectra monitoring the tryptophan (Trp) emission of free BM and in presence of all the surfactants at three different temperatures (288.15 K, 298.15 K, and 308.15 K) reveal the nature of fluorescence quenching of BM in presence of bile salts/surfactants. Time resolved fluorescence studies at room temperature also support to determine the several quenching parameters. The binding constant (Kb) of BM with all the surfactants and free energy of binding (∆G0 of bile salts/surfactants with BM at different temperatures have been calculated exploiting steady state fluorescence technique. It is observed that, the binding of NaC with BM is greater as compared to other surfactants while Stern-Volmer quenching constant (KSV) is found greater in presence of SDBS as compared with others which supports the surface tension and ITC data with the fact that surface activity of surfactant(s) is decreasing with the binding of the surfactants at the core or binding pocket of BM. Circular Dichroism (CD) study shows the stability of secondary structure of BM in presence of NaC and NaDC below C3, while BM lost its structural stability even at very low surfactant concentration of SDDS and SDBS which also supports the more involvement of bile salts in binding rather than surfactants. The molecular docking studies have also been substantiated for better understanding the several experimental investigations interaction of BM with the bile salts/surfactants.

Design of Azomethine based Metalloorganic Complexes and their Binding Interactions with Nucleic Acid and Protein

Mononuclear nickel, copper, and zinc complexes based on salen type (ONNO) donor acting as a tetradentate ligand obtained by the condensation of 4-methyl-o-phenylenediamine and 5-fluoro salicylaldehyde in 1:2 ratios. The compounds are characterized using UV-visible, FT-IR, 1H, 13C-NMR, HR-MS, Elemental (CHN) analyses ESR spectral techniques, and single crystal X-ray diffraction studies (SC-XRD). The binding interactions of the metal complexes with deoxyribonucleic acid (DNA) and bovine serum albumin (BSA) are monitored using UV absorption titration and tryptophan emission quenching techniques, respectively. The complexes possess strong binding propensities with DNA and prefer groove binding, as indicated by the hyperchromic effect. The complexes quench the intrinsic fluorescence of BSA with increasing amounts of the complex concentration in a dose-dependent manner. The gel electrophoresis was carried out against pBR322 to analyze the cleaving ability of the complexes. The results of the NBO (natural bond orbitals) analysis performed in combination with the Time-dependent Density Functional Theory (TD-DFT) calculations provided a good explanation of their electronic and structural characteristics. Further, the predictions of molecular docking studies support the experimental observations. The Gel electrophoresis study reveals the cleaving ability of the complexes by their interactions with DNA. In addition, the cell viability of the complexes is assessed based on MTT assay and the results indicate that they have a good antiproliferative activity only on liver cancer cell-line HepG-2 and does not show cytotoxic effect on normal vero cell lines with 90% viability.

Physico-chemical and structural modifications of caseins in micellar casein isolate induced by pulsed electric field

The potential of pulsed electric field (PEF) to modify the structure of caseins in micellar casein isolate (MCI) was investigated. MCI was PEF-treated at room (RT, 23 °C) and cold (CT, 4 °C) temperatures, using an electric field strength of 16 kV cm−1 for 6 or 31 μs. Conditions simulating elderly digestion, such as the peptide profile released after gastric digestion and bioactivity, were studied. Unfolding of the protein structure was observed in PEF-treated MCI at RT, which led to cleavage points that were more accessible to enzymes. By contrast, fluorescence emission of tyrosine and tryptophan and Raman spectra indicated a reorganization in the configuration of the micelle in PEF-processed at CT. Under these conditions was observed an increase in the number of peptides generated from gastric digestion compared with untreated MCI, especially the bioactive peptides FSDKIAK f(31–45) and MPFPKYPVEP f(124–133), despite a lower overall uptake of bioactive peptides by Caco-2 cells.Industrial relevance: Micellar casein isolate (MCI) is widely utilized for protein fortification (e.g., in fresh dairy products, smoothies, meat products, and clinical nutrition) and for the adjustment of functional properties in food (e.g., viscosity, foaming). The findings of this work provide valuable information about the potential of PEF processing to modify the properties of MCI.

Facile Synthesis of Bis‐Coumarins Using [BCMIM][Cl] Zwitter Ionic Liquid and Their Binding with Bovine Serum Albumin

AbstractHerein, we report a novel synthetic pathway for synthesizing bis‐coumarins using 5 mol% [BCMIM][Cl] ionic liquid via the facile, one‐pot, metal‐free synthesis in aqueous ethanol at room temperature. A series of 24 derivatives were furnished in up to 94% yields in 15 min to 1.5 h and were characterized using different spectroscopic techniques. The photophysical properties of bis‐coumarin derivatives were explored through UV‐Visible and fluorescence spectroscopy. The absorption and emission spectra demonstrate a peak wavelength (λmax), within the range of 297–311 nm and 486–505 nm, respectively. An analogous pattern to the experimental results is seen in density functional calculations. The binding affinities of all the synthesised derivatives with Bovine Serum Albumin (BSA) were determined using AutoDock Vina which exhibited a range from −8.4 to −11.5 kcal mol−1. The computed binding constant (KBSA) by tryptophan (Trp) emission quenching values illustrate strong binding interactions of the selected ligands in the order of 104. The calculated binding affinities were validated by performing molecular dynamics simulations of the best docking pose using GROMACS software over a period of 1 ns for studying the stability of protein‐ligand complexes.

Calcium dependence of both lobes of calmodulin is involved in binding to a cytoplasmic domain of SK channels.

KCa2.1-3 Ca2+-activated K+-channels (SK) require calmodulin to gate in response to cellular Ca2+. A model for SK gating proposes that the N-terminal domain (N-lobe) of calmodulin is required for activation, but an immobile C-terminal domain (C-lobe) has constitutive, Ca2+-independent binding. Although structures support a domain-driven hypothesis of SK gate activation by calmodulin, only a partial understanding is possible without measuring both channel activity and protein binding. We measured SK2 (KCa2.2) activity using inside-out patch recordings. Currents from calmodulin-disrupted SK2 channels can be restored with exogenously applied calmodulin. We find that SK2 activity only approaches full activation with full-length calmodulin with both an N- and a C-lobe. We measured calmodulin binding to a C-terminal SK peptide (SKp) using both composition-gradient multi-angle light-scattering and tryptophan emission spectra. Isolated lobes bind to SKp with high affinity, but isolated lobes do not rescue SK2 activity. Consistent with earlier models, N-lobe binding to SKp is stronger in Ca2+, and C-lobe-binding affinity is strong independent of Ca2+. However, a native tryptophan in SKp is sensitive to Ca2+ binding to both the N- and C-lobes of calmodulin at Ca2+ concentrations that activate SK2, demonstrating that the C-lobe interaction with SKp changes with Ca2+. Our peptide-binding data and electrophysiology show that SK gating models need deeper scrutiny. We suggest that the Ca2+-dependent associations of both lobes of calmodulin to SKp are crucial events during gating. Additional investigations are necessary to complete a mechanistic gating model consistent with binding, physiology, and structure.

Solubility and Thermal Stability of Thermotoga maritima MreB.

The basis of MreB research is the study of the MreB protein from the Thermotoga maritima species, since it was the first one whose crystal structure was described. Since MreB proteins from different bacterial species show different polymerisation properties in terms of nucleotide and salt dependence, we conducted our research in this direction. For this, we performed measurements based on tryptophan emission, which were supplemented with temperature-dependent and chemical denaturation experiments. The role of nucleotide binding was studied through the fluorescent analogue TNP-ATP. These experiments show that Thermotoga maritima MreB is stabilised in the presence of low salt buffer and ATP. In the course of our work, we developed a new expression and purification procedure that allows us to obtain a large amount of pure, functional protein.

Multifarious analytical capabilities of the UV/Vis protein fluorescence in blood plasma

Autofluorescence of blood plasma has been broadly considered as a prospective disease screening method. However, the assessment of such intrinsic fluorescence is mostly phenomenological, and its origin is still not fully understood, complicating its use in the clinical practice. Here we present the detailed evaluation of analytical capabilities, variability, and formation of blood plasma protein fluorescence based on the open dataset of excitation-emission matrices measured for ∼300 patients with suspected colorectal cancer, and our supporting model experiments. Using high-resolution size-exclusion chromatography coupled with comprehensive spectral analysis, we demonstrate, for the first time, the dominant role of HSA in the formation of blood plasma fluorescence in the visible spectral range (excitation wavelength >350 nm), presumably caused by its oxidative modifications. Furthermore, the diagnostic value of the tryptophan emission, as well as of the tyrosine fluorescence and visible fluorescence of proteins is shown by building a tree-based classification model that uses a small subset of physically interpretable fluorescence features for distinguishing between the control group and cancer patients with >80% accuracy. The obtained results extend current understanding and approaches used for the analysis of blood plasma fluorescence and pave the way for novel autofluorescence-based disease screening methods.

Determination of the boundary lipids of sticholysins using tryptophan quenching

Sticholysins are α-pore-forming toxins produced by the sea-anemone Stichodactyla helianthus. These toxins exert their activity by forming pores on sphingomyelin-containing membranes. Recognition of sphingomyelin by sticholysins is required to start the process of pore formation. Sphingomyelin recognition is coupled with membrane binding and followed by membrane penetration and oligomerization. Many features of these processes are known. However, the extent of contact with each of the different kinds of lipids present in the membrane has received little attention. To delve into this question, we have used a phosphatidylcholine analogue labeled at one of its acyl chains with a doxyl moiety, a known quencher of tryptophan emission. Here we present evidence for the contact of sticholysins with phosphatidylcholine lipids in the sticholysin oligomer, and for how each sticholysin isotoxin is affected differently by the inclusion of cholesterol in the membrane. Furthermore, using phosphatidylcholine analogs that were labeled at different positions of their structure (acyl chains and headgroup) in combination with a variety of sticholysin mutants, we also investigated the depth of the tryptophan residues of sticholysins in the bilayer. Our results indicate that the position of the tryptophan residues relative to the membrane normal is deeper when cholesterol is absent from the membrane.

Characterization of [Ru(bpy)2(diamine)]2+ complexes and their DNA binding and cleavage, BSA interaction, cytotoxic, and anticancer mechanistic properties

The ruthenium(II) complexes of the type [Ru(bpy)2(diamine)](PF6)21–3, where diamine is ethylenediamine (en, 1) or N,N-dimethylethylenediamine (dmen, 2) and N,N-diethyl-ethylenediamine (deen, 3) and bpy is 2,2′-bipyridine has been isolated and characterized. The X-ray crystal structures of 1 and 2 reveal that the compounds possess a distorted octahedral geometry composed of two amine nitrogen atoms of the diamine ligand (1, en; 2, dmen) and four nitrogen atoms of the two bpy ligands, which selectively forms a “cis-α” (2) or “cis-β” (1) isomer. The 1H and 13C NMR spectral data of the complexes disclose the nature of metal-ligand bonding, conformations of the chelate rings, and absolute configurations, which indicate that the two bpy ligands maintain their tendency to form geometrical isomers even in solution. They exhibit dπ → π* MLCT transitions, show a ligand-based radical anion (3MLCT) luminescence, and display one quasi-reversible and one non-reversible metal-based oxidative process corresponding to RuII/RuIII and RuIII/RIV redox couples respectively. They interacted with calf thymus (CT) DNA and the mode of interaction was found to be groove binding, as determined by various spectral and redox techniques. Upon interaction with CT DNA 1–3 show a decrease in current and less positive shift in the formal potential in the differential pulse voltammograms suggesting that they are involved in electrocatalytic guanine oxidation. Interestingly, 1–3 alters the DNA superhelicity upon binding with supercoiled pUC19 plasmid DNA with a reductant. Further, the quenching of the tryptophan emission of BSA in the presence of 1–3 is static. The binding is mainly entropy-driven and hydrophobic forces played a major role. The DNA and protein binding affinity and the DNA cleavage activity is in the order 2 > 3 > 1, and the higher DNA and protein binding affinity and DNA cleavage activity of 2 and 3 illustrates the strong involvement of the methyl (dmen) and ethyl (deen) groups in the hydrophobic interaction with the DNA and protein. Furthermore, the ruthenium(II) complexes, 2 and 3 exhibit moderate cytotoxicity against human breast adenocarcinoma MCF-7 and human cervical carcinoma HeLa (IC50: 48 h, 28–45 μM) cells, whereas 2 displays the best activity (IC50: 48 h, 28 μM) for MCF-7 cancer cells, which is better than that of cisplatin. Both the complexes displayed excellent selectivity by being relatively inactive against the Madin-Darby bovine kidney (MDBK) normal cells with selectivity index (SI) values ranging from 5.6 to 8.9. They block the cell cycle progression of MCF-7 cells in the G1 (2) and S (3) phases. FACSVerse analyses are suggestive of reactive oxygen species (ROS) generation and apoptotic cell death induced by 2 and 3. The induction of apoptosis in MCF-7 cells was shown by Annexin V with propidium iodide (PI) and 4’,6-diamidino-2-phenylindole (DAPI) staining methods.

Identification of bacteria by poly-aromatic hydrocarbon biosensors.

Human health is consistently threatened by different species of pathogenic bacteria. To fight the spread of diseases, it is important to develop rapid methods for bacterial identification. Over the years, different kinds of biosensors were developed for this cause. Another environmental risk is poly-aromatic hydrocarbons (PAHs) that may be emitted from industrial facilities and pollute environmental water and soil. One of the methods for their purification is conducted by the addition of bacteria that can degrade the PAHs, while the bacteria can be filtrated at the end of the process. Although many studies reported monitoring of the PAHs degradation by fluorescence, not much attention was dedicated to studying the influence of the PAHs on the intrinsic fluorescence of the degrading bacteria. In this work, we apply synchronous fluorescence (SF) measurements to study the ability of the 5 PAHs: 9-Antracene carboxylic acid (9ACA), Pyrene, Perylene, Pentacene, and Chrysene to interact with bacteria and change its fluorescence spectra. We show that upon incubation of each PAH with the bacterium E. coli, only the 2 PAHs 9ACA and Perylene cause an intensity decrease in the emission at λ = 300-375nm, which derives from the emission of tyrosine and tryptophan (TT). Also, we show that upon incubation of 9ACA and Perylene with 5 different pathogenic bacteria, the intensity increase or decrease in the TT emission is unique to each bacterial species. Based on this observation, we suggest that the PAHs 9ACA and Perylene can be utilized as biosensors for bacterial identification.

Syntheses, structures, and bioactivities evaluation of three transition metal complexes with 1,2,4-triazole carboxylic derivative

Reactions of 2-{[5-(Pyridin-2-yl)-4H-1,2,4-triazol-3-yl]sulfanyl}acetic acid (H2L) with manganese(II), zinc(II) and cadmium(II) acetate in CH3OH/H2O mixture result in the corresponding metal complexes. All the obtained complexes were characterized by IR spectroscopy, elemental analyses, ESI-MS, and single-crystal X-ray diffraction. Structural analyses reveal the dinuclear complexes with the compositions [Mn2L2(CH3OH)2(H2O)2]·2CH3OH (1), [Zn2L2(CH3OH)2(H2O)2]·2CH3OH (2), and 1D chain structure [CdL(H2O)]n (3) in all complexes, H2L is doubly deprotonated (L2-) and serves as equilibrium anion. The carboxylate acts as monodentate ligand in 1 and 2, While μ1,1-bridging ligand in 3. The measurement of Jack Bean urease inhibitory activity showed that 1 and 3 exhibited considerable inhibitory effects for Jack Bean urease with IC50 Values of 12.4 and 2.7 μM, respectively. In addition, bioactive complexes 1 and 3 can effectively bind to bovine serum albumin (BSA), which was assessed by tryptophan emission quenching measurement

In vitro and in silico assessment of antitumor properties and biomolecular binding studies for two new complexes based on NiII bearing k2N,S-donor ligands

This work deals with two new molecule-based materials, namely NiII-complexes of general formulae [Ni(L1)2] (Ni1) and [Ni(L2)2] (Ni2), where L1 = trans-cinnamaldehyde-N(4)-methyl thiosemicarbazone and L2 = trans-cinnamaldehyde-N(4)-ethyl thiosemicarbazone, as potential antitumor agents. Both compounds were characterized by elemental analysis, molar conductivity and spectroscopic techniques (FTIR and NMR). Their molecular structures were obtained by single-crystal X-ray diffraction analysis. Each one crystallizes in a monoclinic space group P 21/c, also the asymmetric unit comprises of one NiII ion located on an inversion centre and one anionic ligand, which acts as a κ2N,S-donor affording a five-membered metallaring. The compounds were screened against two selected tumour cell lines (MCF-7 and A549) and non-tumour fibroblasts cell line (MRC-5) via MTT assays. In both tumour cells, all compounds exhibited higher cytotoxicity than the control drug (cisplatin). The IC50 values ranges of 3.70 – 41.37 μM and 1.06 – 14.91 μM were found for MCF-7 and A549, respectively. Importantly, all of them were less toxicity than cisplatin in MRC-5 with SI values ranged at 11.80 – 86.60. The red blood cell (RBC) assay revealed Ni2 as non-toxic due to its reduced haemolytic effect (0‐–9% at 1‐–10 μM). The DNA binding was investigated through a combination of spectrophotometric absorption and emission titrations, electrophoresis, and circular dichroism experiments. As a result, these metal complexes were not able to strongly binding to DNA (Kb values ~104 mol L‐−1) but suggesting groove-binding interactions. The scavenging ability of them towards 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical was also evaluated in this work, but no important antioxidant behaviour was detected. Further, the interaction of Ni1 and Ni2 to human serum albumin (HSA) was explored by quenching of tryptophan emission, warfarin competitive assay, and molecular docking protocols. The HSA binding analyses indicated good affinity of both complexes to Sudlow site I (Kb values ⁓103 mol L−1).

Combined Quantum-Classical Simulation of Photoinduced Electronic Density Redistribution from Biopolymer Segments to Photochromic Probes

The mechanism of fluorescence quenching of the human serum albumin by transferring the energy of the photoinduced electronic excitation from the single tryptophan residue in the structure to the nitrospiropyran donor introduced into its environment is studied by the hybrid computer simulation, including the classical molecular dynamics and the semi-empirical photo-physical calculations for generating the statistical spectra of tryptophan emission and spectra of nitrospiropyran absorption. The probability of the electronic excitation redistribution between the donor and the acceptor is estimated, followed by the photochromic conversion of nitrospiropyran to the merocyanine form, which is readily identifiable due to a significant shift of the longwave absorption band and can be treated as a luminescence detector of the ongoing photoprocesses. The mechanisms of the energy transfer between nonequilibrium fragments in typical combinations of their complex are considered in detail. The general scheme and technical specifics of modeling the optical spectra are illustrated using a simple system of the anthracene molecule in argon. A discussion of several other advanced hybrid approaches of the classical methods in combination with the quantum-mechanical calculations, developed at different theoretical levels and applied in the current computational molecular spectroscopy, is presented.

Synthesis and Characterization of Serendipitous Dioxovanadates and Their DNA/BSA Interaction Studies and In Vitro Cytotoxic activity

In the current study, we have synthesized and characterized two, hitherto unreported, bis (N,N) donor dioxovanadium(V) complexes based on 1,10-phenanthroline (complex 1) and 2,2′-bipyridyl (complex 2) ligands with 3,5-dinitrosalicylic acid counter anion. The structure of the complexes are confirmed by single crystal X-ray crystallography. The binding interactions of the metal complexes with deoxyribonucleic acid (DNA) and bovine serum albumin (BSA) have been investigated by UV absorption titration and tryptophan emission quenching methods respectively. The complexes quench the intrinsic fluorescence of BSA with increasing amount of the complex concentration. Furthermore, the cytotoxic effect of the complexes against cervical cancer cell lines (HeLa) has been ascertained by MTT assay. The complexes are found to show good DNA/BSA binding ability. Based on the results obtained, it has been observed that the complexes bind preferably to groove as implicated by hyperchromic effect with increasing amount of the DNA. The binding constants (Kb) of the complexes 1 and 2 are 9.02 × 103 M−1 and 2.13 × 103 M−1 respectively. The quenching constants (KBSA) and quenching rate constant (Kq) of the complexes 1 & 2 are 0.1075 × 106 M−1 & 0.076 × 106 M−1 and 1.075 × 1013 M−1S−1 and 0.76 × 1013 M−1S−1, respectively.

A rapid method to quantify casein in fluid milk by front-face fluorescence spectroscopy combined with chemometrics

Casein in fluid milk determines cheese yield and affects cheese quality. Traditional methods of measuring casein in milk involve lengthy sample preparations with labor-intensive nitrogen-based protein quantifications. The objective of this study was to quantify casein in fluid milk with different casein-to-crude-protein ratios using front-face fluorescence spectroscopy (FFFS) and chemometrics. We constructed calibration samples by mixing microfiltration and ultrafiltration retentate and permeate in different ratios to obtain different casein concentrations and casein-to-crude-protein ratios. We developed partial least squares regression and elastic net regression models for casein prediction in fluid milk using FFFS tryptophan emission spectra and reference casein contents. We used a set of 20 validation samples (including raw, skim, and ultrafiltered milk) to optimize and validate model performance. We externally tested another independent set of 20 test samples (including raw, skim, and ultrafiltered milk) by root mean square error of prediction (RMSEP), residual prediction deviation (RPD), and relative prediction error (RPE). The RMSEP for casein content quantification in raw, skim, and ultrafiltered milk ranged from 0.12 to 0.13%, and the RPD ranged from 3.2 to 3.4. The externally validated error of prediction was comparable to the existing rapid method and showed practical model performance for quality-control purposes. This FFFS-based method can be implemented as a routine quality-control tool in the dairy industry, providing rapid quantification of casein content in fluid milk intended for cheese manufacturing.

Dispersion and Interaction of Charged Fluorescent Dyes in Protein-Polymer Surfactant-based Non-Aqueous Liquid.

Viscous, non-aqueous liquid comprising stoichiometric conjugates of polymer surfactant-bovine serum albumin (PSpBSA) is used as a host matrix for the dispersion of chemically distinct hydrophilic dyes. Using a combination of bright field polarized optical microscopy and fluorescence spectroscopy, we investigate the dispersion of dry and powdered cationic (Rhodamine 6G; Rh6G) and anionic (Fluorescein; FL) dyes in the PSpBSA liquid at room temperature. As the dyes disperse and dissolve in the PSpBSA liquid, it results in a pronounced increase in emission intensity of the former. Interestingly, a shift from 571 to 582 nm is observed in the emission maxima of Rh6G as it disperses in the PSpBSA solvent. Whilst no such red shift is found for the Rh6G dispersion in the aqueous solutions of either native BSA or polymer-surfactant conjugated BSA, a similar shift occurs when Rh6G is dispersed in neat polymer-surfactant (PS), suggesting the interaction of the dye with the PS chains. In the case of anionic FL, no shift is observed in its emission maximum as it disperses in the PSpBSA liquid. Furthermore, within 120 minutes of FL dispersion in the PSpBSA liquid, we observe a ≈26 % decrease in the tryptophan emission intensity (λexc. =285 nm; λemi. =330 nm) of BSA, which could be attributed to both static and dynamic quenching. Our findings provide a proof of concept of an alternative non-aqueous solvent matrix which can dissolve and disperse charged fluorescent dyes, provide suitable binding sites, and show substantial photoluminescence. Thus, it can be envisaged for utilization as an alternative solvent medium for lasing dyes and related applications.

Both Lobes of Calmodulin Bound to KCa2.2 Respond to Ca2+

An increase in intracellular Ca2+ opens KCa2.2 Ca2+-activated K+-channels (SK) to conduct K+ down its electrochemical gradient. SK activation requires bound calmodulin proteins to decode dynamic Ca2+ fluctuations in a cell. Bilobal calmodulin has four Ca2+ binding sites. A common model proposes that just the N-terminal domain (N-lobe) of calmodulin binds Ca2+ to open SK, while an immobile C-terminal domain (C-lobe) has constitutive, Ca2+-independent binding. Such a mechanism is compatible with x-ray and EM structures of intact SK channels and fragments. While molecular structures provide snapshots of the channels in different conformations with and without calcium, gating is a dynamic process involving several different conformations, calcium binding to calmodulin, interactions between calmodulin and the channel and channel opening. We measured SK activity using inside-out patch recordings from “calmodulin-disrupted” SK channels where calcium-dependent activation can be restored by exogenously applied full-length calmodulin. Isolated N and C lobes have little ability to activate SK. We measured calmodulin binding to a C-terminal SK2 peptide (SKp) using both composition-gradient multi-angle light scattering and tryptophan emission spectra. While they do not rescue activation, isolated lobes do bind to a SK cytoplasmic domain with high affinity. Consistent with earlier models, N-lobe binding to SKp is stronger in Ca2+, and C-lobe binding affinity is strong regardless of Ca2+. However, a native tryptophan in SKp is sensitive to Ca2+ binding to either the N- or C-lobe of calmodulin at Ca2+ concentrations that activate SK. Our results show that the calmodulin C-lobe bound to SK is Ca2+ sensitive and that both lobes of calmodulin bind SK in Ca2+-dependent states governed by the different properties of each lobe.

In vitro digestibility, structural and functional properties of Moringa oleifera seed proteins

The aim of this work was to compare the structural and functional properties of Moringa oleifera seed albumin and globulin with those of the isoelectric pH-precipitated protein isolate (ISO). The 0.5 M NaCl extract of defatted Moringa flour was dialyzed against water to give water-soluble albumin (ALB) and water-insoluble globulin (GLO). The three protein products were evaluated for in vitro protein digestibility, functional properties and polypeptide composition while structural conformations were obtained using surface hydrophobicity (So), intrinsic fluorescence and circular dichroism (CD). Results showed that ALB had the most exposed number of hydrophobic groups with So of 946.6 when compared to 7.8 for GLO and 50.4 for ISO. The GLO had the highest protein digestibility while ALB and GLO had least gelation concentration of 0.8%, which is significantly (p < 0.05) lower than the 2.2% for the ISO. At pH 3.0, all the proteins showed tryptophan and tyrosine emission peaks; increases in pH led to disappearance of the tyrosine peak in some of the samples. Gel electrophoresis under reducing conditions suggest the presence of disulfide bonds in the three protein products. CD data indicate GLO as having more β-sheet conformation while the α-helix content varied depending on the pH. At 10, 20 and 40 mg/mL protein concentrations, foaming capacity was comparable in all the samples but the emulsifying properties carried out at 20, 25 and 50 mg/mL showed that ISO and ALB had significantly (p < 0.05) higher emulsifying ability than the GLO.

Quenching of tryptophan fluorescence by colloidal Cu2S nanoparticles through static and dynamic modes under different solution pH

The study of the interaction between biomolecules and nanoparticles (NPs) is interesting since the dimensions of the two overlap to some extent. Herein, the interaction of an essential amino acid, tryptophan, with Cu2S NPs was investigated by using various spectroscopic techniques. The 220 nm absorption band of tryptophan was found to be blue-shifted by 2 and 5 nm at pH 6 and 10.6 respectively. The binding of tryptophan with the Cu2S NPs at pH 10.6 via its indole chromophore was established by the Fourier transform infrared (FTIR) spectroscopic measurements. The fluorescence emission of tryptophan was quenched by the semiconductor NPs. The fluorescence quenching was found to be dynamic in nature at pH 6. However, the static mode of quenching was found to be operative at pH 10.6. The binding constants and thermodynamic parameters were calculated for the tryptophan-Cu2S NPs complex at pH 10.6.