scholarly journals Antagonistic binding of substrates to 3-phosphoglycerate kinase monitored by the fluorescent analogue 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate

1994 ◽  
Vol 301 (3) ◽  
pp. 885-891 ◽  
Author(s):  
M Vas ◽  
A Merli ◽  
G L Rossi
Keyword(s):  
Dissociation Constants ◽  
Equilibrium Dialysis ◽  
Fluorescence Emission ◽  
Phosphoglycerate Kinase ◽  
Structural Data ◽  
Blue Shift ◽  
Competitive Inhibitor ◽  
Intensity Increase ◽  
Fluorescence Emission Spectrum ◽  
Reciprocal Effect

The analogue of ATP, 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate (TNP-ATP), binds tightly to pig muscle 3-phosphoglycerate kinase. A dissociation constant Kd of 0.0095 +/- 0.0015 mM was determined by fluorimetric titration on the basis of 1:1 stoichiometry. TNP-ATP is a strong competitive inhibitor towards MgATP and MgADP with a Ki of 0.008 +/- 0.001 mM for both substrates. It is also a mixed-type inhibitor towards 3-phosphoglycerate with similar inhibition constants. Binding of TNP-ATP to 3-phosphoglycerate kinase is accompanied by a tenfold intensity increase and a blue shift of about 20 nm in its fluorescence emission spectrum and a shift of the pK of its trinitrophenyl group towards a more acidic pH. These findings suggest that the negatively charged trinitrophenyl group of TNP-ATP significantly contributes to the binding of the analogue. By stepwise replacement of the fluorescent TNP-ATP, the dissociation constants (Kd) for ADP and MgADP binding were determined and found to be 0.78 +/- 0.08 and 0.048 +/- 0.006 mM respectively, which are consistent with the values previously determined by equilibrium dialysis [Molnár and Vas (1993) Biochem J. 293, 595-599]. In similar competitive-titration experiments, ATP and MgATP did not completely substitute for TNP-ATP. For the fraction of the analogue that could be substituted, the dissociation constants for MgATP and ATP were estimated to be 0.27 +/- 0.09 and 0.33 +/- 0.15 mM respectively, close to the values determined by equilibrium dialysis. Using the same method, a significant weakening of binding of both (Mg)ADP and (Mg)ATP could be detected in the presence of 3-phosphoglycerate: their respective Kd values became 0.34 +/- 0.04 and 0.51 +/- 0.22 mM. The reciprocal effect, i.e. weakening of 3-phosphoglycerate binding in the presence of the nucleotide substrates, has been observed previously [Vas and Batke (1984) Eur. J. Biochem. 139, 115-123]. Similarly, a much weaker binding of (Mg)ATP could be observed in the presence of 1,3-bisphosphoglycerate (Kd = 2.30 +/- 0.68 mM). The possible reason for the mutual weakening of substrate binding is discussed in the light of the available structural data.

A novel ratiometric and reversible fluorescent probe based on naphthalimide for the detection of Al3+ and pH with excellent selectivity

2020 ◽  
Vol 44 (8) ◽  
pp. 3261-3267 ◽  
Author(s):  
Zhuo Li ◽  
Weihong Chen ◽  
Liuyan Dong ◽  
Yan Song ◽  
Ronghang Li ◽  
...  
Keyword(s):  
Emission Spectrum ◽  
Fluorescent Probe ◽  
Fluorescence Probe ◽  
Ph Value ◽  
Fluorescence Emission ◽  
Blue Shift ◽  
Living Cells ◽  
Fluorescence Emission Spectrum ◽  
Excellent Selectivity

A novel ratiometric and reversible fluorescence probe was designed and synthesized. The fluorescent probe recognizes Al3+ to cause a blue shift in the fluorescence emission spectrum. Fluorescence probe can be used as a sensor to detect Al3+ in living cells and Zebra fishes. The probe can detect the pH value when the alkalinity range was 8–10.

Superficial disposition of the N-terminal region of the surfactant protein SP-C and the absence of specific SP-B–SP-C interactions in phospholipid bilayers

2001 ◽  
Vol 359 (3) ◽  
pp. 651-659 ◽  
Author(s):  
Inés PLASENCIA ◽  
Antonio CRUZ ◽  
Cristina CASALS ◽  
Jesús PÉREZ-GIL
Keyword(s):  
Emission Spectrum ◽  
Protein Interactions ◽  
Fluorescence Emission ◽  
Resonance Energy ◽  
Fold Increase ◽  
Blue Shift ◽  
Terminal Segment ◽  
Phospholipid Bilayers ◽  
Aqueous Environment ◽  
Fluorescence Emission Spectrum

A dansylated form of porcine surfactant-associated protein C (Dns-SP-C), bearing a single dansyl group at its N-terminal end, has been used to characterize the lipid–protein and protein–protein interactions of SP-C reconstituted in phospholipid bilayers, using fluorescence spectroscopy. The fluorescence emission spectrum of Dns-SP-C in phospholipid bilayers is similar to the spectrum of dansyl-phosphatidylethanolamine, and indicates that the N-terminal end of the protein is located at the surface of the membranes and is exposed to the aqueous environment. In membranes containing phosphatidylglycerol (PG), the fluorescence of Dns-SP-C shows a 3-fold increase with respect to the fluorescence of phosphatidylcholine (PC), suggesting that electrostatic lipid–protein interactions induce important effects on the structure and disposition of the N-terminal segment of the protein in these membranes. This effect saturates above 20% PG molar content in the bilayers. The parameters for the interaction of Dns-SP-C with PC or PG have been estimated from the changes induced in the fluorescence emission spectrum of the protein. The protein had similar Kd values for its interaction with the different phospholipids tested, of the order of a few micromolar. Cooling of Dns-SP-C-containing dipalmitoyl PC bilayers to temperatures below the phase transition of the phospholipid produced a progressive blue-shift of the fluorescence emission of the protein. This effect is interpreted as a consequence of the transfer of the N-terminal segment of the protein into less polar environments that originate during protein lateral segregation. This suggests that conformation and interactions of the N-terminal segment of SP-C could be important in regulating the lateral distribution of the protein in surfactant bilayers and monolayers. Potential SP-B–SP-C interactions have been explored by analysing fluorescence resonance energy transfer (RET) from the single tryptophan in porcine SP-B to dansyl in Dns-SP-C. RET has been detected in samples where native SP-B and Dns-SP-C were concurrently reconstituted in PC or PG bilayers. However, the analysis of the dependence of RET on the protein density excluded specific SP-B–Dns-SP-C associations.

scholarly journals Intra-domain communication between the N-terminal and DNA-binding domains of the androgen receptor: modulation of androgen response element DNA binding

2005 ◽  
Vol 34 (3) ◽  
pp. 603-615 ◽  
Author(s):  
Jacqueline Brodie ◽  
Iain J McEwan
Keyword(s):  
Androgen Receptor ◽  
Dna Binding ◽  
Fluorescence Emission ◽  
Progesterone Receptors ◽  
Fluorescence Emission Spectrum ◽  
Response Element ◽  
Amino Terminal ◽  
Binding Domains ◽  
Receptor Modulation

The androgen receptor (AR) is a ligand-activated transcription factor that recognises and binds to specific DNA response elements upon activation by the steroids testosterone or dihydrotestosterone. In vitro, two types of response element have been characterised - non-selective elements that bind the androgen, glucocorticoid and progesterone receptors, and androgen receptor-selective sequences. In the present study, the allosteric effects of DNA binding on the receptor amino-terminal domain (NTD) were studied. Binding to both types of DNA response element resulted in changes in the intrinsic fluorescence emission spectrum for four tryptophan residues within the AR-NTD and resulted in a more protease-resistant conformation. In binding experiments, it was observed that the presence of the AR-NTD reduced the affinity of receptor polypeptides for binding to both selective and non-selective DNA elements derived from the probasin, PEM and prostatin C3 genes respectively, without significantly altering the protein–base pair contacts. Taken together, these results highlight the role of intra-domain communications between the AR-NTD and the DNA binding domain in receptor structure and function.

scholarly journals Interaction of rice (Oryza sativa) lectin with N-acetylglucosaminides. Fluorescence studies

1985 ◽  
Vol 229 (3) ◽  
pp. 687-692 ◽  
Author(s):  
F Tabary ◽  
J P Frénoy
Keyword(s):  
Oryza Sativa ◽  
Binding Sites ◽  
Wheat Germ ◽  
Equilibrium Dialysis ◽  
Blue Shift ◽  
Association Constants ◽  
Fluorescence Studies ◽  
Saccharide Binding ◽  
Binding Enthalpy

The interaction of lectin isolated from rice (Oryza sativa) embryos with N-acetylglucosaminides was studied by equilibrium dialysis and fluorescence. Equilibrium dialysis with 4-methylumbelliferyl-(GlcNac)2 showed that rice lectin (Mr 38000) contains four equivalent saccharide-binding sites. Addition of the N-acetylglucosaminides GlcNac, (GlcNac)2 and (GlcNac)3 enhanced the intrinsic fluorescence of rice lectin and this was accompanied by a 10nm blue-shift of its maximum fluorescence with (GlcNac)2 and (GlcNac)3. These changes in intensity allowed determination of the association constants, which increased with the number of saccharide units: at 20 degrees C, Ka = (1.3 +/- 0.1) X 10(3), (5.1 +/- 0.4) X 10(4) and (2.6 +/- 0.1) X 10(5) M−1 for GlcNac, (GlcNac)2 and (GlcNac)3 respectively. The binding enthalpy, delta H0, for the three glucosaminides were very low and ranged from −12.1 to −20.6 kJ X mol-1. The results are compared with those obtained with wheat-germ agglutinin, another GlcNac-specific gramineaous lectin.

scholarly journals Combining Hollow Core Photonic Crystal Fibers with Multimode, Solid Core Fiber Couplers through Arc Fusion Splicing for the Miniaturization of Nonlinear Spectroscopy Sensing Devices

Fibers ◽  
2018 ◽  
Vol 6 (4) ◽  
pp. 77 ◽  
Author(s):  
Hanna Stawska ◽  
Maciej Popenda ◽  
Elżbieta Bereś-Pawlik
Keyword(s):  
Photonic Bandgap ◽  
Average Power ◽  
Fluorescence Emission ◽  
Nonlinear Spectroscopy ◽  
Fluorescence Emission Spectrum ◽  
Solid Core ◽  
Hollow Core ◽  
Fluorescence Excitation ◽  
Photonic Bandgap Fiber ◽  
Sensing Applications

The presence of fiber optic devices, such as couplers or wavelength division multiplexers, based on hollow-core fibers (HCFs) is still rather uncommon, while such devices can be imagined to greatly increase the potential of HCFs for different applications, such as sensing, nonlinear optics, etc. In this paper, we present a combination of a standard, multimode fiber (MMF) optic coupler with a hollow core photonic bandgap fiber through arc fusion splicing and its application for the purpose of multiphoton spectroscopy. The presented splicing method is of high affordability due to the low cost of arc fusion splicers, and the measured splicing loss (SL) of the HCF-MMF splice is as low as (0.32 ± 0.1) dB, while the splice itself is durable enough to withstand a bending radius (rbend) of 1.8 cm. This resulted in a hybrid between the hollow core photonic bandgap fiber (HCPBF) and MMF coupler, delivering 20 mW of average power and 250-fs short laser pulses to the sample, which was good enough to test the proposed sensor setup in a simple, proof-of-concept multiphoton fluorescence excitation-detection experiment, allowing the successful measurement of the fluorescence emission spectrum of 10−5 M fluorescein solution. In our opinion, the presented results indicate the possibility of creating multi-purpose HCF setups, which would excel in various types of sensing applications.

The Possibility of Predicting Prolongation of the Pharmacological Action of Biologically Active Polymeric Substances

2012 ◽  
Vol 39 (11) ◽  
pp. 21-27 ◽  
Author(s):  
L.I. Shal'nova ◽  
N.A. Lavrov ◽  
A.F. Nikolaev
Keyword(s):  
Dissociation Constants ◽  
Equilibrium Dialysis ◽  
Physiological Activity ◽  
Pharmacological Action ◽  
Physicochemical Characteristics ◽  
Biologically Active ◽  
Organic Bases ◽  
Physiologically Active Substances ◽  
Biological Methods ◽  
The Relationship

The physicochemical properties and the formation, stability, and dissociation constants of complexes of poly acids with organic bases possessing physiological activity were determined by methods of potentiometric titration, conductometry, and equilibrium dialysis, and by biological methods. The relationship between the level of physicochemical characteristics of complexes and the possibility of predicting the prolonged pharmacological (antimicrobial, anaesthetising) action of physiologically active substances in complexes with polymers was established.

Characterization of hydrocarbon fluid inclusions by infra-red and fluorescence microspectrometry

1990 ◽  
Vol 54 (375) ◽  
pp. 311-324 ◽  
Author(s):  
Nicole Guilhaumou ◽  
Nathalie Szydlowskii ◽  
Bernard Pradier
Keyword(s):  
Fluid Inclusions ◽  
Aromatic Hydrocarbons ◽  
Fluorescence Emission ◽  
Liquid Hydrocarbon ◽  
Fluorescence Emission Spectrum ◽  
Hydrocarbon Fluid ◽  
Liquid Phases ◽  
Infra Red ◽  
Aliphatic And Aromatic Hydrocarbons

AbstractLiquid-hydrocarbon-bearing fluid inclusions have often been described associated with petroleum occurrences and diagenetic sediments. Infra-red microspectrometry allows characterization of fluid inclusions greater than 20 µm by establishing the presence of aliphatic and aromatic hydrocarbons as well as associated H2O, CO2 and CH4. Semi-quantitative analyses have been made by focussing on gaseous and liquid phases separately. Some CH2/CH3 and CO2/CH4 ratios have been determined by this method.Fluorescence microspectrometry permits precise measurements of the fluorescence emission spectrum of chromophore-bearing organic phases (essentially aromatic hydrocarbons) in fluid inclusions greater than 10 µm. Such a spectrum is a function of both the gross composition of the trapped oil and its thermal history.Both of these methods lead to the in situ characterization of hydrocarbon fluid inclusions. They are useful in providing a quantifiable distinction between different oil generations trapped during mineral growth in diagenetic and epigenetic minerals.

scholarly journals The Synthesis and Application of Nitrogen-Doped Graphene Quantum Dots on Brilliant Blue Detection

2019 ◽  
Vol 2019 ◽  
pp. 1-9 ◽  
Author(s):  
Li Jin ◽  
Ying Wang ◽  
Fengkai Yan ◽  
Jianpo Zhang ◽  
Fangli Zhong
Keyword(s):  
Quantum Dots ◽  
Emission Spectrum ◽  
Graphene Quantum Dots ◽  
Fluorescence Emission ◽  
Fluorescence Decay ◽  
Brilliant Blue ◽  
Fluorescence Emission Spectrum ◽  
Nitrogen Doped ◽  
Nitrogen Doped Graphene ◽  
Doped Graphene

Nitrogen-doped graphene quantum dots had been successfully synthesized and characterized by using transmission electron microscope, X-ray photoelectron spectroscopy, absorbance spectrum, fluorescence emission spectrum, and fluorescence decay curve. TEM results indicated that the diameters of the as-prepared nitrogen-doped graphene quantum dots were in the range of 2 - 5 nm and the lattice space is about 0.276 nm; Raman spectrum result indicated that there were two characteristic peaks, generally named D (~1408 cm−1) and G (~1640 cm−1) bands; both TEM and Raman spectrum results indicated that the as-synthesized product was graphene quantum dots. Deconvoluted high resolution XPS spectra for C1s, O1s, and N1s results indicated that there are -NH-, -COOH, and -OH groups on the surface of nitrogen-doped graphene quantum dot. Fluorescence emission spectrum indicated that the maximum fluorescence emission spectrum of nitrogen-doped graphene quantum dots was blue shift about 30.1 nm and the average fluorescence decay time of nitrogen-doped graphene quantum dots increased about 2 ns, compared with graphene quantum dots without doping of nitrogen. Then, the as-prepared nitrogen-doped graphene quantum dots were used to quantitatively analyze brilliant blue based on the fluorescent quenching of graphene quantum dots, and the effect of pH and reaction time on this fluorescent quenching system was also obtained. Under selected condition, the linear regression equations were F0/F=0.0087 (brilliant blue) + 0.9553 and F0/F=0.01205 (brilliant blue) + 0.6695, and low detection limit was 3.776 μmol/L (3.776 nmol/mL). Once more diluted N-GQDs (0.05 mg/mL) were used, the low detection limit could reach 94.87 nmol/L. Then, temperature-dependent experiment, absorbance spectra, and dynamic fluorescence quenching rate constant were used to study the quenching mechanism; all results indicated that this quenching process was a static quenching process based on the formation of complex between nitrogen-doped graphene quantum dots and brilliant blue through hydrogen bond. Particularly, this method was used to quantitatively analyze the wine sample, of which results have a high consistence with the results of the spectrophotometric method; demonstrating this fluorescence quenching method could be used in practical sample application.

Conformational Flexibility In Fibrinogen

1981 ◽  
Author(s):  
Roy R Hantgan
Keyword(s):  
Relaxation Time ◽  
Polyacrylamide Gel Electrophoresis ◽  
Fluorescence Emission ◽  
Structural Data ◽  
Rotational Relaxation ◽  
Average Lifetime ◽  
Rigid Sphere ◽  
Thrombin Activation ◽  
Hydrodynamic Data ◽  
Rotational Relaxation Time

The structure of fibrinogen in solution has been investigated by the technique of steady state fluorescence polarization. Factor XIII a has been employed to catalyze the incorporation of approximately 3 moles dansylcadaverine per mole fibrinogen without disruption of its structure or function. Two γ-chain and one α-chain crosslink acceptor sites have been labelled, according to SDS-polyacrylamide gel electrophoresis. Light scattering and electron microscopy verify that this derivative forms normal fibrin upon thrombin activation; the modified protein is 93% clottable. The fluorescence emission maxiumum is shifted to 500 nm (vs. 540 nm for dansylcadaverine) and a substantial increase in fluorescence intensity is noted. Measurements of the fluorescent lifetime, by a phase shift technique, show an average lifetime of 14.5 ±2.5 nanoseconds (vs. 3 ns for dansylcadaverine). The rotational relaxation time, determined from a Perrin plot of polarization vs. temperature, is 165 ±13 ns. No change in rotational relaxation time was found in 0.005 M CaCl2 or at high ionic strength. The limiting polarization (extrapolated to infinite viscosity) agrees with that determined with dansylcadaverine fibrin (coarse gel conditions) as well as with the vlaue measured in 75% glycerol at 0°C. These controls confirm that overall macromolecular motion, not probe rotation,is being measured.The measured rotational relaxation time for dansylcadaverine fibrinogen is substantially shorter than values calculated for an anhydrous rigid sphere (300 ns) or a prolate ellipsoid (1000 ns), as well as other models based on hydrodynamic data. These results indicate that the rotating unit is smaller than the entire fibrinogen molecule, i.e. that the structure of fibrinogen in solution is flexible. This observation is consistent with earlier polarization data reported for dansylchloride labelled fibrinogen, as well as structural data indicating fibrinogen to be a multidomain protein, whose globular portions are connected by helical chains.

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