Room-temperature tryptophan phosphorescence of proteins of isolated human erythrocyte membranes

Results of phosphorescent analysis of proteins incorporated into an erythrocyte membrane in the native state and after depleting it of peripheral proteins of both the spectrin–actin network (bands 1, 2, and 5) and bands 2.1-2.3, 4.1, 4.2, and 6 are presented. We also investigated the ability of peripheral proteins extracted from membranes to produce room-temperature tryptophan phosphorescence in solution. V. M. Mazhul’ (Deceased). Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 76, No. 2, pp. 267–272, March–April, 2009.     

Phenylalanine fluorescence and phosphorescence used as a probe of conformation for cod parvalbumin

The fluorescence emission and triplet absorption properties of phenylalanine in cod fish parvalbumin type II, a protein that contains no Trp or Tyr, was examined in the time scale ranging from nanoseconds to microseconds at 25°C in aqueous buffer (pH 7.0). In the presence of Ca(II), the decay of fluorescence gave two lifetimes (5.9 and 53 ns) and the triplet lifetime was 425 s. Upon removal of Ca, the fluorescence intensity decreased to values approaching that for free Phe, while the longest fluorescence decay component was 17 ns. At the same time, the decay of triplet showed complex nonexponential kinetics with decay rates faster than in the presence of Ca. Quenching and denaturation analyses suggest that the Phe's are present in a hydrophobic environment in the Ca-bound protein but that the Ca-free protein is relatively unstructured. It is concluded that Phe luminescence in proteins is sensitive to conformation and that the long lifetime of Phe excited states needs to be considered when studying its photochemistry in proteins.     

Adsorption of firefly luciferase at interfaces studied by total internal reflection fluorescence spectroscopy

The adsorption of luciferase onto silica surfaces was studied by total internal reflection fluorescence (TIRF) spectroscopy. Two model surfaces were used: hydrophilic and hydrophobic silica. Luciferase adsorbed differently on these two surfaces. Initial kinetics of luciferase adsorption onto the hydrophilic surface showed that luciferase adsorbs over an adsorption energy barrier of 3 kT The quantum yield of luciferase fluorescence decreased at the hydrophilic silica surface, which indicated that the protein conformation was altered during adsorption. Luciferase adsorption onto the hydrophobic silica surface proceeded with a small adsorption energy barrier and the fluorescence efficiency of adsorbed protein remained unchanged after adsorption. The affinity of luciferase for luciferin was measured using quenching of luciferase fluorescence with luciferin. The binding constant of the adsorbed luciferase-luciferin complex at the hydrophilic silica surface was two orders of magnitude smaller than the respective binding constant in the solution. Adsorbed luciferase showed an absence of ATP-dependent visible luminescence, indicating that the adsorbed enzyme was not active at either of the two silica surfaces.     

Stopped-flow fluorescence studies of the interaction of a mutant form of cytochrome b5 with lipid vesicles

Cytochrome b₅ binds spontaneously to lipid vescles and also self-associates in aqueous solution. Two mutant proteins have been generated, one has a self-association constant which is less than that of the native protein, while the other has a larger self-association constant. All three proteins have Trp in the membrane-binding domain but as aqueous solutions of these proteins contain differing amounts of monomeric protein, the kinetics of fluorescence enhancement, when the proteins are mixed with lipid vesicles, are complex. Similar complex kinetics are seen when the Trp are quenched by the addition of bromolipid vesicles. The mutant which has Trp 108 and 112 both replaced by Leu does not self-associate and shows monoexponential stopped-flow fluorescence kinetics. Identical rate constants are seen with this mutant for fluorescence enhancement by POPC and fluorescence quenching by three bromolipids with bromines at the 6,7-, 9,10-, and 11,12-positions of thesn-2 acyl chain. This rate constant is only 1% of the calculated collisional rate constant and it is suggested that the reduced rate is caused by a reduction in the number of productive collisions rather than by a slow rate of penetration of the membrane-binding domain into the bilayer.     

X-ray excited fluorescence and phosphorescence of CaSO4: Sm phosphors

Calcium sulphate phosphors activated with samarium as an impurity with various percentage composition have been prepared and their fluorescence rise and phosphorescence decay behaviours have been studied. The growth curve is investigated to study the density of traps. The process of filling of traps during x-ray excitation is revealed. The decay curves are analysed to study the distribution of traps, the nature of decay and kinetics involved in the luminescence process. The concentration dependence of fluorescence emission of the phosphors is also discussed.     

Interaction of a phosphatidylcholine derivative of 1,6-diphenyl-1,3,5-hexatriene (DPH) with intact living cells: Steady-state fluorescence polarization and phase fluorometry studies

The potential interest of DPH-PC was checked with a macrophagic cell line (P388D1). The uptake of DPH-PC was associated with a rapid increase in both fluorescence intensity and a slow decrease in anisotropy values. A flow cytometry comparative study with DPH revealed in both cases the existence of two cell subpopulations with different labeling levels. The analysis of fluorescence decay of DPH-PC showed two components. The fractional intensity of the main component (9.7 ns) is higher than 92%. The Lorentzian distribution of the main lifetime presents an important homogeneity. The observation that an increase in temperature induced a decrease in steady state anisotropy values but did not affect the lifetime suggests that the anisotropy variations effectively reflect modifications in the cohesion of probe micro-surroundings. A transmembrane diffusional phenomenon of a fraction of fluorescent phospholipids (205) was suggested by a study with a nonpermeant membrane quencher. The transmembrane diffusion was confirmed by extraction of the phospholipid analog with fatty acid free BSA. The use of inhibitors of endogenous phospholipase A2 showed a progressive hydrolysis of the fluorescent phospholipid. Nevertheless, the hydrolysis can be neglected in the case of short term interactions with cells (<30 min). Therefore, it can be assumed that DPH-PC can be used as a membrane probe.     

Davydov splitting and two-photon excitation of cadmium tungstate (CdWO4) single crystal phosphorescence

Phosphorescence characteristics of CdWO₄ excited by one-photon (λ = 308 nm) and two-photon (λ = 570–590 nm) processes were measured. A Davydov splitting of 120 ± 20 cm⁻¹ was obtained in the phosphorescence spectra, suggesting a diffusion coefficient of about 1.2 × 10⁻² cm² s⁻¹, and a diffusion length of about 3.1 × 10⁻⁴ cm for the room temperature measured lifetime of 8μs. The phosphorescence quantum efficiency was less than 2% at low temperatures (only 0.25% at room temperature), indicating that the dominant decay mechanism was radiationless. The radiative lifetime was thus estimated as 1–2 ms. The two-photon phosphorescence excitation is characterized by an absorption cross-section of the order of 10⁻⁴⁹cm⁴s.     

Recombination Prolonged Luminescence of Indole and Tryptophan in a Solution at Room Temperature

Prolonged luminescence of tryptophan and indole in liquid solutions of different polarity has been registered in a microsecond time-scale at room temperature; by nature it differs from phosphorescence or delayed fluorescence of P and E types. The data presented suggest that the observed prolonged luminescence is caused by recombination of the radicals formed upon UV irradiation of indole chromophores by laser pulses.     

Quenching interactions and nonexponential decay: tryptophan 138 of bacteriophage T4 lysozyme

Site-directed mutagenesis has been used to prepare variants of bacteriophage T4 lysozyme that contain only one tryptophan residue at position 138 and to change the residues in the immediate environment of this buried residue. Replacement of glutamine-105 by alanine results in a 2.7-fold increase in fluoresence quantum yield and converts the fluorescence decay from a highly nonexponential form to a single-exponential decay. This is atributed to electron transfer quenching of tryptophan-138 fluorescence by glutamine-105. Replacemeent of alanine-146 by threonine results in a 1.6-fold decrease in fluorescence intensity, indicating enhanced quenching by glutamine-105; replacement of glutamine-105 by alanine in this species results in a 5-fold in crease in fluorescence intensity. The interpretation of the nonexponential decay of the glutamine-105-containing species is discussed in terms of reversibility of the quenching process.     

Dynamics ofLens culinaris agglutinin studied by red-edge excitation spectra and anisotropy measurements of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) and of tryptophan residues

The fluorescence of 2-p-toluidinylnaphthalene-6-sulfonate bound toLens culinaris agglutinin and of the Trp residues of the protein was investigated. Red-edge excitation spectra and steady-state anisotropy as a function of temperature indicate that the TNS is bound rigidly. Red-edge excitation spectra, steady-state anisotropy as a function of sucrose and anisotropy decay experiments performed on Trp residues fluorescence prove that the internal fluorophore presents residual motion independent of the global rotation of the protein. Fluorescence anisotropy decay allows to calculate the rotational correlation time (351 ps) of this local motion. Quenching resolved emission anisotropy with iodide gives values equal to 0.257 and 0.112 for the anisotropies of the buried and the surface Trp residues, respectively. This result indicates that the Trp residues present at the surface of the protein have important local motions compared to those embedded in the protein matrix. The results obtained from TNS and Trp residues indicate that the agglutinin has different dynamic domains.     

Delayed fluorescence and phosphorescence of tris-(8-hydroxyquinoline)aluminum (Alq3) and their temperature dependence

This paper reports on delayed luminescence measurements of tris-(8-hydroxyquinoline)aluminum (Alq₃) after optical excitation; these identify two bands in the emission spectrum: delayed fluorescence and phosphorescence. This is shown for different crystalline phases and for evaporated films. The assignment of the low-energy band to the phosphorescence is confirmed by lifetime measurements, and triplet energies of the meridional isomer in the -phase and the facial isomer in the δ-phase are determined from the well-resolved vibronic progressions of the phosphorescence as 2.11±0.1 and 2.16±0.1 eV, respectively. Lifetimes of the delayed fluorescence and the phosphorescence are determined for a temperature range from 6 to 150 K, and we measured the temperature dependence of the delayed photoluminescence spectra.     

Fluorescence intensity and anisotropy decays of the intrinsic tryptophan emission of hemoglobin measured with a 10-GHz fluorometer using front-face geometry on a free liquid surface

We measured the intensity and anisotropy decays of the intrinsic tryptophan emission from hemoglobin solutions obtained using a 10-GHz frequency-domain fluorometer and a specially designed cuvette which allows front-face excitation on a free liquid surface. The cuvette eliminates reflections and stray emissions, which become significant for low-intensity fluorescence such as in hemoglobin. Three lifetimes are detectable in the subnanosecond range. The average lifetime of hemoglobin emission is ligand dependent. The measured values of average lifetimes are 91, 174, and 184 ps for deoxy-, oxy-, and carboxyhemoglobin, respectively. Fluorescence anisotropy decays of oxy-, deoxy-, and carbonmonoxyhemoglobin can be fitted with up to three correlation times. When three components are used, the floating initial anisotropyr _o is, in each case, higher than the steady-state anisotropy of tryptophan in vitrified solution. For deoxy hemoglobin it is close to 0.4. The data are consistent with an initial loss of anisotropy from 0.4 to about 0.3 occurring in the first 2 ps.     

Optical and photoelectron spectroscopic studies of alkyl-passivated silicon nanoparticles

We have performed the optical and photoelectron spectroscopic studies of alkyl-passivated Si nanoparticles synthesized by a solution route. The alkyl-passivated Si nanoparticle with mean diameter less than about 2 nm exhibits a strong ultraviolet-blue photoluminescence. Furthermore, we have directly investigated their electronic structures in the vicinity of Fermi level by means of valence-band photoemission measurements using synchrotron radiation. From these results, the detailed optical properties and electronic structures of alkyl-passivated Si nanoparticles are discussed.     

Absorption and fluorescence spectra of hybrid silicon-organic materials in solutions and films

The spectroscopic characteristics of hybrid organic-inorganic films and solutions of ethoxysilicon-containing amidophosphate ligands in acetonitrile have been investigated. Inhomogeneous broadening of the absorption and fluorescence spectra which is characterized by the spectral dependence of the fluorescence and fluorescence excitation bands on the excitation and registration wavelength, respectively, is associated with the presence of a set of -(Si-O)_n- siloxane structures with various terminal fragments in both the films and solutions. The established general spectral rules are discussed with allowance for the characteristic features of the electronic structure of silicon-organic compounds containing an oxygen atom the unshared electron pair of which ensures the delocalization of the electron density over the siloxane chain.Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 71, No. 6, pp. 788–792, November–December, 2004.This revised version was published online in April 2005 with a corrected cover date.     

lin-Benzo-ATP and-ADP: Versatile fluorescent probes for spectroscopic and biochemical studies

lin-Benzo-adenine nucleotides can act not only as probes for fluorescence studies but also as structural active site probes for enzymes. To understand the basic properties oflin-benzo-ATP and-ADP, protolysis and Mg²⁺ and Ca²⁺, binding are investigated between pH 6.2 and pH 8.5 by spectrophotometric and spectrofluorometric titrations. Based on a reaction model, a set of equilibrium constants is determined which is consistent with all available experimental results. The pK values of the Mg²⁺ and Ca²⁺ complex oflin-benzo-ATP in the chosen medium are 4.6 and 4.1, respectively, and those for the corresponding diphosphate are 3.1 and 2.8, respectively. Fluorescence and absorption spectra are reported.This is a peer-reviewed conference proceeding article from the Third Conference on Methods and Applications of Fluorescence Spectroscopy, Prague, Czech Republic, October 18–21, 1993.     

Fluorescence-quenching studies and temperature dependence of fluorescence quantum yield, decay time and intersystem crossing activation energy of TPB

Fluorescence quenching of 1, 1, 4, 4-tetraphenyl-1, 3-butadiene (TPB) by aniline has been carried out at room temperature (298 K) to understand the role of quenching mechanisms. The study has been carried out by both steady state (in different solvents) and by transient method (in cyclohexane). The Stern-Volmer plot has been found to be linear for all the solvents studied. The probability of quenching per encounter ‘p’ is determined in all the solvents and is found to be less than unity. It is found that, the activation energy E_a (E′_a) is greater than the activation energy of diffusion, E_d. The results obtained by the transient method infer that the thermally assisted intersystem crossing, a non-radiative deactivation process from S₁ to T₂ is responsible for observed decrease in quantum yield and lifetime. Hence, from both the methods it can be concluded that quenching mechanism is not solely due to the material diffusion, but there is also contribution from the activation energy.