scholarly journals Spectral studies on the cadmium-ion-binding properties of bovine brain S-100b protein

1991 ◽  
Vol 276 (1) ◽  
pp. 13-18 ◽  
Author(s):  
H Donato ◽  
R S Mani ◽  
C M Kay
Keyword(s):  
Absorption Band ◽  
Fluorescence Intensity ◽  
Fluorescence Emission ◽  
Bovine Brain ◽  
Spectral Studies ◽  
Tyrosine Residue ◽  
Difference Spectroscopy ◽  
Model Complexes ◽  
Emission Maximum ◽  
Fluorescence Measurements

The effect of Cd2+ binding on bovine brain S-100b protein was studied using c.d. u.v. difference spectroscopy and fluorescence measurements. At pH 7.5, S-100b protein binds two Cd2+ ions per monomer with a Kd value of 3 x 10(-5) M. Addition of Cd2+ resulted in perturbing the single tyrosine residue (Tyr17) in the protein as indicated by u.v. difference spectroscopy and aromatic c.d. measurements. In the presence of Cd2+, the tyrosine residue moves to a more non-polar environment, since a red shift was observed in the u.v. difference spectrum. When the protein was excited at 278 nm, the tyrosine fluorescence emission maximum was centred at 306 nm. Cd2+ addition resulted in an increase in intrinsic fluorescence intensity. Fluorescence titration with Cd2+ indicated the protein binds Cd2+ with a Kd value of 3 x 10(-5) M. 2-p-Toluidinylnaphthalene-6-sulphonate-labelled protein, when excited at 345 nm, had a fluorescence emission maximum at 440 nm. Addition of Cd2+ to labelled protein resulted in a 5-fold increase in fluorescence intensity accompanied by a 5 nm blue shift in the emission maximum, suggesting that the probe, in the presence of Cd2+, moves to a hydrophobic domain. U.v. difference spectroscopic studies indicated a unique Cd2(+)-binding site on the protein, since Cd2+ addition yielded a large positive absorption band in the 240 nm region that is not found with either Ca2+ or Zn2- ions. Similar absorption bands have been observed in Cd-protein complexes such as Cd-metallothionein [Vasak, Kagi & Hill (1981) Biochemistry 20, 2852-2856] and also in model complexes of Cd2+ with 2-mercaptoethanol. This absorption band is believed to arise as a result of charge-transfer transitions between the thiolate and Cd2+. Of the two Cd2- -binding sites on the beta-chain, one must be located at the N-terminal end near the single tyrosine residue, since Cd2- and Zn2+ produced similar effects on the intrinsic protein fluorescence. The other Cd2+ site which is unique to Cd2+ must be Cys84, located at the C-terminal end.

scholarly journals Purification and spectral studies on the Ca2+-binding properties of 67 kDa calcimedin

1989 ◽  
Vol 259 (3) ◽  
pp. 799-804 ◽  
Author(s):  
R S Mani ◽  
C M Kay
Keyword(s):  
Fluorescence Intensity ◽  
Binding Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Fluorescence Emission ◽  
Spectral Studies ◽  
Affinity Column ◽  
Protein Fluorescence ◽  
Hydrophobic Region ◽  
Emission Maximum

The 67 kDa calcimedin, isolated by using a phenyl-Sepharose affinity column followed by DEAE-cellulose and gel-filtration chromatographies, was homogeneous by the criterion of SDS/polyacrylamide-gel electrophoresis. In non-SDS gels, the protein moved faster in the presence of EDTA, suggesting that Ca2+ binding affects its mobility in a manner similar to other Ca2+-binding proteins such as calmodulin and S-100 proteins. The 67 kDa protein underwent a conformational change upon binding Ca2+, as revealed by u.v. difference spectroscopy and near-u.v. c.d. measurements. Tryptophan and tyrosine residues were perturbed upon Ca2+ binding, moving to a more non-polar environment in the presence of Ca2+. Upon excitation of the protein at 280 nm, the fluorescence emission maximum was centered around 325 nm, suggesting that the tryptophan residues are located in a fairly hydrophobic region. Ca2+ addition did not induce a significant change in the intrinsic protein fluorescence intensity at 325 nm. Addition of Ca2+ to the 67 kDa protein labelled with 2-p-toluidinylnaphthalene-6-sulphone (TNS) resulted in a 25% increase in fluorescence intensity, accompanied by a blue shift of the emission maximum from 442 to 432 nm. Hence, the probe in the presence of Ca2+ moves to a more non-polar microenvironment, like calmodulin and other Ca2+-binding proteins. Fluorescence titration with Ca2+ using TNS-labelled protein revealed one class of binding site, with a Kd value of 2 x 10(-5) M.

Halothane and Isoflurane Alter the Calcium sup 2+ Binding Properties of Calmodulin

1995 ◽  
Vol 83 (1) ◽  
pp. 120-126. ◽  
Author(s):  
Aaron Levin ◽  
Thomas J. J. Blanck
Keyword(s):  
Binding Protein ◽  
Fluorescence Emission ◽  
Volatile Anesthetic ◽  
Volatile Anesthetics ◽  
Bovine Brain ◽  
Hill Coefficient ◽  
Excitation Wavelength ◽  
Binding Properties ◽  
Fluorescence Measurements ◽  
The Hill

Background Ca2+ plays an important role in signal transduction and anesthetic mechanisms. To date, no one has observed a direct effect of volatile anesthetics on a Ca(2+)-binding protein. We therefore examined the effects of halothane and isoflurane on the Ca(2+)-binding properties of bovine brain calmodulin. Methods The fluorescence emission of calmodulin was obtained over a range of Ca2+ concentrations (10(-7)-10(-4)M) in the presence and absence of halothane and isoflurane. The intrinsic tyrosine fluorescence of calmodulin was measured at an excitation wavelength of 280 nm and an emission wavelength of 320 nm. Fluorescence measurements were carried out in 50 mM hydroxyethylpiperazineethane sulfonic acid, 100 mM KC1, and 2 mM ethyleneglycol-bis-(beta-aminoethyl ether) tetraacetic acid at pH 7.0 and 37 degrees C. Experiments were performed in polytetrafluorethylene-sealed cuvettes so that the volatile anesthetic concentrations remained constant. The titration data were analyzed in two ways. The data were fit to the Hill equation by using nonlinear regression analysis to derive the Hill coefficient and the dissociation constant. The data were also analyzed by two-way analysis of variance with multiple comparisons to determine statistically significant effects. Volatile anesthetic concentrations were measured by gas chromatography. Results The presence of volatile anesthetics altered the Ca(2+)-binding affinity of calmodulin in a dose-dependent fashion. At 0.57% (0.25 mM) halothane and 1.7% (0.66 mM) isoflurane, the affinity of calmodulin for Ca2+ relative to control was decreased. However, at higher concentrations of both anesthetics, the affinity for Ca2+ was increased. When the volatile anesthetics were allowed to evaporate from the experimental solutions, the observed rightward shift of the calmodulin-Ca2+ binding curve for Ca2+ at low concentrations of the anesthetics returned to the control position. The leftward shift seen at high concentrations of the anesthetics was irreversible after evaporation of 8.7% (3.3 mM) isoflurane and 5.7% (2.5 mM) halothane. Conclusions These data demonstrate a complex interaction of two hydrophobic volatile anesthetics with calmodulin. A biphasic effect was observed both for halothane and for isoflurane. Calmodulin, an EF-hand Ca(2+)-binding protein, undergoes a conformational shift when binding Ca2+, exposing several hydrophobic residues. These residues may be sites at which the anesthetics act.

A fluorochrome from aniline blue: structure,synthesis and fluorescence properties

1982 ◽  
Vol 35 (12) ◽  
pp. 2571 ◽  
Author(s):  
NA Evans ◽  
PA Hoyne
Keyword(s):  
Critical Micelle Concentration ◽  
Cationic Surfactant ◽  
Fluorescence Intensity ◽  
Fluorescence Emission ◽  
Aniline Blue ◽  
Fluorescence Properties ◽  
Hexadecyltrimethylammonium Bromide ◽  
Commercial Sample ◽  
Emission Maximum ◽  
Structure Synthesis

A fluorochrome has been isolated in analytically pure form from a commercial sample of the triaryl-methane dye aniline blue. Its structure has been shown to be sodium 4,4'-[carbonylbis(benzene-4,1-diyl)bis(imino)]bisbenzenesulfonate by spectroscopic means and confirmed by synthesis. Its fluorescence emission, which is markedly solvent-dependent, is 150 times greater in butan-1-ol than in water (however, the wavelength of the emission maximum is not altered significantly). In the presence of a cationic surfactant, hexadecyltrimethylammonium bromide, the fluorescence intensity reaches a maximum at approximately the critical micelle concentration of the surfactant.

scholarly journals Ca2+ and Zn2+-binding properties of nitrated S-100b protein from bovine brain

1986 ◽  
Vol 238 (3) ◽  
pp. 715-719 ◽  
Author(s):  
R S Mani ◽  
C M Kay
Keyword(s):  
Conformational Changes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Difference Spectrum ◽  
Bound State ◽  
Bovine Brain ◽  
Tyrosine Residue ◽  
Difference Spectroscopy ◽  
Native Protein ◽  
Binding Properties ◽  
Helical Content

The single tyrosine residue in S-100b protein was nitrated by treatment with tetranitromethane in 0.1 M-Tris/HCl buffer, pH 8.0, containing 2 mM-EDTA. The nitrated protein did not differ significantly in secondary structure from its native unmodified counterpart, as revealed by far-u.v. c.d. measurements. The effect of Ca2+ on the modified protein was different from that on the native protein, e.g. addition of Ca2+ resulted in a loss of helical content from 55 to 47% with the native protein whereas Ca2+ had no significant effect on the gross conformation of the nitrated derivative. Near-u.v. c.d. studies also indicated a very minimal effect on the tyrosine residue and this was also reflected in the u.v.-absorption difference spectrum. Polyacrylamide-gel electrophoresis in the absence of SDS showed the nitrated S-100b to move faster in the presence of EDTA compared with the calcium-bound state, suggesting that the modified protein does bind Ca2+ although it does not undergo a major conformational change in response to Ca2+ addition. In contradistinction, Zn2+ binding was not influenced by nitration, as demonstrated by aromatic c.d. and u.v.-difference spectroscopy. It is clear from this study that the single tyrosine residue in S-100b is critical to sense the Ca2+-induced conformational changes in the protein.

scholarly journals Isolation, characterization and metal-ion-binding properties of the α-subunit from S-100a protein

1986 ◽  
Vol 237 (3) ◽  
pp. 757-764 ◽  
Author(s):  
I K M Leung ◽  
R S Mani ◽  
C M Kay
Keyword(s):  
Conformational Change ◽  
Metal Ion ◽  
Polyacrylamide Gel ◽  
Alpha Subunit ◽  
Ion Binding ◽  
Difference Spectroscopy ◽  
Metal Ion Binding ◽  
Emission Maximum ◽  
Fluorescence Measurements ◽  
Fluorescence Titration

The brain-specific S-100 protein is a mixture of two predominant components, S-100a and S-100b, with subunit compositions of alpha beta and beta beta respectively. In the present study, the alpha-subunit, isolated from S-100a by using anion-exchange chromatography in the presence of 8 M-urea, was homogeneous by the criteria of SDS/polyacrylamide-gel, urea/SDS/polyacrylamide-gel and non-SDS/polyacrylamide-gel electrophoresis. The alpha-subunit underwent a conformational change upon binding Ca2+ and Zn2+ at pH 7.5, as revealed by u.v. difference spectroscopy, c.d. and fluorescence measurements. Far-u.v. c.d. studies indicated the apparent alpha-helical content to fall when the protein bound either Ca2+ or Zn2+. Addition of Ca2+ to the alpha-subunit resulted in exposing to the solvent the single tryptophan residue and one or more tyrosine and phenylalanine residues. Zn2+ induced only a small conformational change, and among the aromatic chromophores only tyrosine residues were affected to a small extent. Ca2+ was able to bind to the alpha-subunit in the presence of Zn2+, and the two metal-ion-binding sites appeared to be different. When the apoprotein was excited at 280 nm, the fluorescence emission maximum was located at 337 nm. In the presence of Ca2+, the emission maximum occurred at 340 nm and was accompanied by a nearly 25% increase in fluorescence intensity. Fluorescence titration with Ca2+ at pH 7.5 revealed only one class of binding site, with a Kd value of 1.26 × 10(-4) M. The effect of K+ on the protein was slightly antagonistic to that of Ca2+, as indicated by u.v. difference spectroscopy and fluorescence titration.

scholarly journals Sponge-derived Ageladine A affects the in vivo fluorescence emission spectra of microalgae

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0242464
Author(s):  
Carolin Peter ◽  
Silke Thoms ◽  
Florian Koch ◽  
Franz Josef Sartoris ◽  
Ulf Bickmeyer
Keyword(s):  
Natural Products ◽  
Fluorescence Intensity ◽  
Crucial Role ◽  
Fluorescence Emission ◽  
Emission Spectra ◽  
Fluorescence Emission Spectra ◽  
Tisochrysis Lutea ◽  
Emission Maximum ◽  
Ph Dependent

In several marine hosts of microalgae, fluorescent natural products may play an important role. While the ecological function of these compounds is not well understood, an interaction of these molecules with the photosynthesis of the symbionts has been suggested. In this study, the effect of Ageladine A (Ag A), a pH-dependent fluorophore found in sponges of the genus Agelas, on microalgal fluorescence was examined. The spectra showed an accumulation of Ag A within the cells, but with variable impacts on fluorescence. While in two Synechococcus strains, fluorescence of phycoerythrin increased significantly, the fluorescence of other Synechococcus strains was not affected. In four out of the five eukaryote species examined, chlorophyll a (Chl a) fluorescence intensity was modulated. In Tisochrysis lutea, for example, the position of the fluorescence emission maximum of Chl a was shifted. The variety of these effects of Ag A on microalgal fluorescence suggests that fluorophores derived from animals could play a crucial role in shaping the composition of marine host/symbiont systems.

scholarly journals INTERACTION OF COPPER OXIDE NANOPARTICLES WITH BOVINE SERUM ALBUMIN BY SPECTROSCOPIC STUDIES

2018 ◽  
Vol 10 (5) ◽  
pp. 35
Author(s):  
Suja Abraham ◽  
Vellaichamy Parthasarathy
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Fluorescence Intensity ◽  
Bovine Serum ◽  
Structural Changes ◽  
Fluorescence Emission ◽  
Average Particle ◽  
Time Resolved ◽  
Cuo Nps ◽  
Fluorescence Measurements

Objective: Since structural changes of adsorbed protein are necessary for cellular uptake of nanoparticles (NPs) it is of prime importance to know about structural changes of bovine serum albumin (BSA) when it interacts with CuO NPs–a potential new antitumor drug.Methods: CuO NPs prepared by sol-gel technique were characterized by x-ray diffraction (XRD) and tunneling electron microscope (TEM) techniques. The conformational changes induced by CuO NPs on BSA were studied by various spectroscopic techniques such as steady state and time-resolved fluorescence measurements. The changes in fluorescence emission parameters such as fluorescence intensity, fluorescence emission maximum and lifetimes of fluorescent residues in BSA were studied.Results: XRD analysis showed the average particle size as 32 nm. The TEM micrograph showed particles of different size varying from 10 to 45 nm. Fluorescence quenching was confirmed due to a decrease in fluorescence intensity of CuO NPs–BSA complex. The analysis of lifetime measurements indicated BSA contained two tryptophan (trp) residues that fluoresced in different environments. Static quenching mechanism was confirmed by time-resolved measurements when BSA interacted with CuO NPs.Conclusion: Minor structural changes of BSA protein were observed during the interaction studies.

Efficient control of dye molecules fluorescence in polydopamine via strong optical coupling in a SPASER nanoparticle

2021 ◽  
Author(s):  
Pavel Melentiev ◽  
Boris Khlebtsov ◽  
Anton Gritchenko ◽  
Denis Kudryavtsev ◽  
Igor Ivanov ◽  
...  
Keyword(s):  
Viral Particle ◽  
Direct Detection ◽  
Fluorescence Emission ◽  
Radiative Properties ◽  
Environment Interaction ◽  
Optical Coupling ◽  
Dye Molecules ◽  
Sensing Applications ◽  
Fluorescence Measurements ◽  
Optical Modes

Abstract One of the main approaches of design and manipulation of the quantum emitters’ radiative properties is based on proper control of optical modes of the surrounding quantum emitter environment, leading to, in its ultimate case, to a strong regime of the emitter - environment interaction. In this Letter, we present the realization of a mesoscopic physical system (Au SPASER nanoparticle with polydopamine shell containing dye molecules Cy 7.5) in which the strong optical coupling regime allows: (i) to get rid of dye molecules quenching, (ii) to suppress photobleaching of dye molecules and (iii) to get a great enhancement of dye molecules fluorescence. We have reduced the rate of fluorescence quenching of dye molecules in PDA by 1000 times and increased the fluorescence emission rate of dye molecules by 30 time, thus preparing bright, nanoscale and biocompatible fluorescent probes suitable for bio-sensing applications. As an example of practical use of the probe, we demonstrate direct detection of single SARS-CoV-2 viral particle via fluorescence measurements of the probes attached to the viral particle through the antibodies.

scholarly journals Single occupancy spectroelectrochemistry of freely diffusing flavin mononucleotide in zero-dimensional nanophotonic structures

2015 ◽  
Vol 184 ◽  
pp. 101-115 ◽  
Author(s):  
Lawrence P. Zaino ◽  
Dane A. Grismer ◽  
Donghoon Han ◽  
Garrison M. Crouch ◽  
Paul W. Bohn
Keyword(s):  
Electron Transfer ◽  
Single Molecule ◽  
Light Emission ◽  
Fluorescence Emission ◽  
Flavin Mononucleotide ◽  
Wide Field ◽  
Potential Sweep ◽  
Potential Control ◽  
Fluorescence Measurements ◽  
Electron Transfer Properties

Zero-mode waveguides (ZMW) have the potential to be powerful confinement tools for studying electron transfer dynamics at single molecule occupancy conditions. Flavin mononucleotide contains an isoalloxazine chromophore, which is fluorescent in the oxidized state (FMN) while the reduced state (FMNH2) exhibits dramatically lower light emission, i.e. a dark-state. This allows fluorescence emission to report the redox state of single FMN molecules, an observation that has been used previously to study single electron transfer events in surface-immobilized flavins and flavoenzymes, e.g. sarcosine oxidase, by direct wide-field imaging of ZMW arrays. Single molecule electron transfer dynamics have now been extended to the study of freely diffusing molecules using fluorescence measurements of Au ZMWs under single occupancy conditions. The Au in the ZMW serves both as an optical cladding layer and as the working electrode for potential control, thereby accessing single molecule electron transfer dynamics at μM concentrations. Consistent with expectations, the probability of observing single reduced molecules increases as the potential is scanned negative, Eappl < Eeq, and the probability of observing emitting oxidized molecules increases at Eappl > Eeq. Different single molecules exhibit different electron transfer properties as reflected in the position of Eeq and the distribution of Eeq among a population of FMN molecules. Two types of actively-controlled electroluminescence experiments were used: chronofluorometry experiments, in which the potential is alternately stepped between oxidizing and reducing potentials, and cyclic potential sweep fluorescence experiments, analogous to cyclic voltammetry, these latter experiments exhibiting a dramatic scan rate dependence with the slowest scan rates showing distinct intermediate states that are stable over a range of potentials. These states are assigned to flavosemiquinone species that are stabilized in the special environment of the ZMW nanopore.

A multipurpose instrument for quantitative intravital microscopy

1992 ◽  
Vol 73 (1) ◽  
pp. 296-306 ◽  
Author(s):  
A. Toth ◽  
M. E. Tischler ◽  
M. Pal ◽  
A. Koller ◽  
P. C. Johnson
Keyword(s):  
Parenchymal Cell ◽  
Fluorescence Emission ◽  
Repeated Measurements ◽  
Open Architecture ◽  
In Vitro Tests ◽  
Fluorescence Measurements ◽  
Tissue Area ◽  
Microcirculatory Function

An in vivo microscope system has been developed that can measure fluorescence emission and/or light absorption at up to five wavelengths in a tissue area of 18–30 microns diam while imaging adjacent microcirculatory vessels with a video system. The system also incorporates a computer-controlled stage and data acquisition system for rapid and repeated measurements from a number of tissue sites. The tissue area monitored for fluorescence or absorption can be defined further by a confocal arrangement of the microscope optics. Tests of the system for NADH fluorescence measurements show good agreement between the fluorescence at 450 nm and NADH concentration in vitro and in skeletal muscle. The instrument can also be used simultaneously for spectrophotometric determination of O2 saturation and hematocrit in microcirculatory vessels. In vitro tests indicate suitable accuracy for such measurements. The open architecture and modular arrangement of the instrument facilitates its use for a variety of simultaneous measurements of parenchymal cell and microcirculatory function.

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