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At least 19 recordsLinked to original sources
PubMed1977

The use of the phosphorescence microscope for the study of the phosphorescence of various cells.

The phosphorescence microscope (i.e. a microspectrophosphorimeter) was used for recording the phosphorescence of microobjects. The general optical scheme of the reflecting phosphorescence microscope is described. The recordings of the phosphorescence spectra of cells of various types (bacteria, yeast, protozoa and human fibroblasts) were carried out both at room temperature and deep cooling. It will be shown that the phosphorescence spectra of the microorganisms are specific for every species of cells. The changes of the spectral parameters and triplet decay times after X-irradiation of Tetrahymena pyriformis cells were demonstrated. The decay time of phosphorescence of the fibroblasts at low temperature was also obtained.

Animals↗
PubMedDate not supplied

[Study of nucleoside phosphorescence with a phosphorescent microscope].

Phosphorescence of ribonucleosides and desoxyribonucleosides is studied. The phosphorescence spectra of polycrystalline powders of adenosine, guanosine, thymidine, uridine, cytidine, desoxyadenosine, desoxyguanosine and desoxycytidine are recorded at -175 degrees C as well as the decay curves at -175 degrees C. The phosphorescence spectrum of guanosine is also recorded at room temperature. The decay times of phosphorescence of adenosine and cytidine are determined at room temperature too. Obtained values of pi for nucleosides are shorter than those for nucleic acid bases.

Adenosine↗
PubMed2004

Blue phosphorescent Zn(II) and orange phosphorescent Pt(II) complexes of 4,4'-diphenyl-6,6'-dimethyl-2,2'-bipyrimidine.

To investigate the different phosphorescent promoting effects of organic emitters by various metal centers, a new ligand, 4,4'-diphenyl-6,6'-dimethyl-2,2'-bipyrimidine (pmbp), and its Zn(II), Hg(II), and Pt(II) complexes, [Zn(pmbp)(2)](ClO(4))(2)(1), Pt(pmbp)Ph(2)(2), Zn(pmbp)Cl(2)(3), and Hg(pmbp)Cl(2)(4) were synthesized. Their structures were determined by single crystal X-ray diffraction. The zinc complexes 1 and 3 exhibit blue luminescence in the solid state at ambient temperature, but the mercury complex 4 is not luminescent. At 77 K, both pmbp and complex have blue emissions in MeOH solutions, which were demonstrated to be phosphorescence by their long decay lifetime (micros). By comparing the luminescent properties of the free ligand and the complex, we concluded that the phosphorescence of originates from ligand centered pi --> pi* transitions. Complex 2 exhibits orange luminescence both in CH(2)Cl(2) solution at 77 K and in the solid state at ambient temperature, which was assigned to metal-to-ligand [d(M) --> pi*(pmbp)] charge transfer (MLCT). The different origin of luminescence is responsible for the different luminescent color of the Zn(II) and Pt(II) complexes.

Journal Article↗
PubMed1992

Room-temperature phosphorescence from azurin derivatives. Phosphorescence quenching in oxidized native azurin.

The tryptophan phosphorescence from a series of derivatives of Pseudomonas aeruginosa azurin has been monitored at 30 degrees C in pH 8.5 buffer solution. The phosphorescence lifetimes fall in the range of 230-270 ms for deoxygenated solutions of derivatives containing Cd(II), Cu(I), Co(II), Ni(II), Hg(II) or apoazurin. A weak signal with a lifetime of ca 130 ms is observed from solutions of oxidized native azurin, but this component is ascribed to a modified form of azurin in solution, i.e. protein heterogeneity, on the basis of the unique sensitivity to quenching by dioxygen. Aside from this minor component, the tryptophan phosphorescence in the Cu(II) protein appears to be fully quenched. The quenching is assigned an electron-transfer mechanism involving transient reduction of the metal center. The same mechanism is deemed to be responsible for fluorescence quenching in oxidized native azurin as well. These observations are of interest because aromatic groups like tryptophan may be conduits for physiological electron-transfer processes involving the copper center.

Azurin↗
PubMed1994

Heme protein dynamics studied by phosphorescence of an external phosphorescent probe molecule.

The rate constant for quenching, kq, of the phosphorescence of 6-bromo-2-naphthyl sulfate (BNS) by cytochromes c, cytochrome c peroxidase, catalase, and myoglobin has been measured as a function of temperature and solvent viscosity. In aqueous solution at pH 7.0 for cytochromes c and myoglobin the value of kq is nearly equal to the rate constant for diffusional intermolecular contact, which is estimated from the value of kq for microperoxidase-11. For cytochrome c peroxidase and catalase kq is at least 350 times smaller than the rate of diffusional quenching, which shows that quenching of BNS phosphorescence occurs predominantly over the short distance between donor and acceptor. The mechanism for cytochrome c and myoglobin is found not to involve static quenching, deep penetration of BNS into the globin, or unfolding of the protein to allow contact between heme and BNS. It is concluded that quenching occurs by interaction of BNS with the exposed heme edge and by surface insertion of BNS into the protein to a depth sufficient for quenching by the unexposed heme. The effect of rapid-diffusional enhancement on kq is small. From a comparison of the results for the heme proteins, a model emerges that describes cytochrome c and myoglobin as having dynamic surfaces. Sufficient fluctuations persist to allow penetration of polyatomic probe molecules into the protein matrix, but the dynamics and/or interior microenvironment acts to increase resistance with increasing depth of penetration.

Animals↗
PubMed2005

[Comparison of non-protected fluid room temperature phosphorescence and micellar-stabilized room temperature phosphorescence properties of indole-3-butyric acid].

For indole-3-butyric acid (IBA), the non-protected fluid room temperature phosphorescence (NP-RTP) and the fluid room temperature phosphorescence with polymeric dispersant polyethyleneglycol-200 (PEG-200), PEG-400 or non-ionic surfactant Tween-20, Tween-40, Tween-80, Tween-85, Brij35 and emulsifier OP as a medium have been studied in detail. When the surfactants or polymeric dispersants were added to the NP-RTP system, the profile of RTP spectrum was not changed, but the RTP intensity and the pre-irradiation time required to attain a stable RTP signal were increased. Whether the surfactants or polymeric dispersants were added to the system or not, no RTP signal was observed when TlNO3 was used as a heavy atom perturber (HAP), but intense RTP emissions were observed when KI was present as HAP. Recoveries of 95.2%-104% with relative standard deviations of 2.4%-4.0% were obtained in spiked water and soil samples.

Indoles↗
PubMed1998

[Phenomena of supramolecular assembly fluid room temperature phosphorescence (II)--effects of alcohols on room temperature phosphorescence of cyclodextrin/4-iodo-4'-ethyl-biphenyl/bromocyclohexane system].

Beta-cyclodextrin( beta/-CD)can induce 4-iodo-4'-ethyl-biphenyl(IEBP)to emit room temperature phosphorescence(RTP) in the presence of bromocyclohexane (BCH). If butyl alcohol (BuOH) is presented as a fourth component at the same time, RTP intensity of the system is enhanced obviously. Effects of different alcohols, CDs and derivatives of beta-CD on RTP emission of the system are studied. The results show that a ternary host-guest inclusion complex is formed among beta-CD, IEBP and BCH, but the alcohol hydroxyl bonds to the rim of the CD cavities by hydrogen-bonding and the alkyl end of alcohol flips over the two tops of the CD due to hydrophobic interaction force. Accordingly, the alcohol acts as a lid for CD cup, thereby shielding IEBP from oxygen and decreasing the collision quenching of oxygen, and decreasing the micro polarity of the CD cavity and the formation constant of ternary inclusion complex increases. As a result, the RTP of the system is enhanced. As far as we can see that the system studied is a typical supramolecular assembly one and the BuOH acts as a molecular regulation for RTP emitting.

Alcohols↗
PubMed1994

Time-resolved room temperature protein phosphorescence: nonexponential decay from single emitting tryptophans.

The single room temperature phosphorescent (RTP) residue of horse liver alcohol dehydrogenase (LADH). Trp-314, and of alkaline phosphatase (AP), Trp-109, show nonexponential phosphorescence decays when the data are collected to a high degree of precision. Using the maximum entropy method (MEM) for the analysis of these decays, it is shown that AP phosphorescence decay is dominated by a single Gaussian distribution, whereas for LADH the data reveal two amplitude packets. The lifetime-normalized width of the MEM distribution for both proteins is larger than that obtained for model monoexponential chromophores (e.g., terbium in water and pyrene in cyclohexane). Experiments show that the nonexponential decay is fundamental; i.e., an intrinsic property of the pure protein. Because phosphorescence reports on the state of the emitting chromophore, such nonexponential behavior could be caused by the presence of excited state reactions. However, it is also well known that the phosphorescence lifetime of a tryptophan residue is strongly dependent on the local flexibility around the indole moiety. Hence, the nonexponential phosphorescence decay may also be caused by the presence of at least two states of different local rigidity (in the vicinity of the phosphorescing tryptophan) corresponding to different ground state conformers. The observation that in the chemically homogeneous LADH sample the phosphorescence decay kinetics depends on the excitation wavelength further supports this latter interpretation. This dependence is caused by the wavelength-selective excitation of Trp-314 in a subensemble of LADH molecules with differing hydrophobic and rigid environments. With this interpretation, the data show that interconversion of these states occurs on a time scale long compared with the phosphorescence decay (0.1-1.0 s). Further experiments reveal that with increasing temperature the distributed phosphorescence decay rates for both AP and LADH broaden, thus indicating that either 1) the number of conformational states populated at higher temperature increases or 2) the temperature differentially affects individual conformer states. The nature of the observed heterogeneous triplet state kinetics and their relationship to aspects of protein dynamics are discussed.

Alcohol Dehydrogenase↗
PubMed1989

Phosphorescence measurements of calf gamma-II, III, and IV crystallins at 77 and 293 K.

Structural and dynamical features of bovine gamma-crystallin tryptophan residues were investigated by phosphorescence measurements at 77 and 293 K. The low temperature phosphorescence spectra and lifetimes of calf gamma-II, III, and IV crystallins did not reflect heterogeneity among the gamma-crystallins. The 0-0 bands were all at 414 +/- 1 nm and the emission lifetimes were all single-exponential with lifetimes of 5.1, 5.3 and 5.3 +/- 0.3 sec, respectively. In contrast, phosphorescence measurements at room temperature were sensitive to subtle differences in exposure, accessibility, and flexibility of gamma-crystallin tryptophan residues. Thorough deoxygenation allowed for measurement of the normally-quenched room-temperature phosphorescence, and we report the first native phosphorescence measurements of lens crystallins at ambient temperature. The emission maxima for gamma-II, III and IV were 446, 442, and 440 +/- 2 nm, respectively. The intensity decay curves were all non-single exponential, and the decays were fit to a sum of two exponentials with lifetimes of 9.1 and 93 msec (gamma-II), 11 and 75 msec (gamma-III), and 4.2 and 68 msec (gamma-IV), +/- 10%. The components of the gamma-II emission were assigned to the four tryptophans based on X-ray structural information. Quantum yields of the phosphorescence emission were in the ratio of 20:7:1 for gamma-II, III and IV, and comparison of lifetimes and quantum yields suggests that tryptophan rigidity increases in the order gamma-IV less than III less than II. Acrylamide quenching constants for the long-lived components of gamma-II and III were roughly equal, while the short-lived tryptophans of gamma-III were an order of magnitude more accessible than those of gamma-II. The wide range of phosphorescence lifetimes and quenching constants allowed for discrimination of distinct contributions to the phosphorescence emission, and we suggest that room-temperature phosphorescence measurements will be an effective tool for studying conformational changes of lens crystallins.

Animals↗
PubMed1988

Tryptophan phosphorescence of the Ca2+-ATPase of sarcoplasmic reticulum.

Phosphorescence of protein tryptophan was analyzed in sarcoplasmic reticulum vesicles, and in the purified Ca2+ transport ATPase in deoxygenated aqueous solutions at room temperature. Upon excitation with light of 295 nm wavelength, the emission maxima of fluorescence and phosphorescence were at 330 nm and at 445 nm, respectively. The phosphorescence decay was multiexponential; the lifetime of the long-lived component of phosphorescence was approximately equal to 22 ms. ATP and vandate significantly reduced the phosphorescence in the presence of either Ca2+ or EGTA; ADP was less effective, while AMP was without effect. The quenching by ATP showed saturation consistent with the idea that the ATP-enzyme complex had a lower phosphorescence yield. Upon exhaustion of ATP, the phosphorescence returned to starting level. Significant quenching of phosphorescence with a decrease in phosphorescence lifetime was also caused by NaNO2, methylvinyl ketone and trichloroacetate, without effect on ATPase activity; this quenching did not show saturation and was therefore probably collisional in nature.

Adenosine Triphosphate↗
PubMed1997

Time-resolved room temperature tryptophan phosphorescence in proteins.

The application of luminescence, primarily fluorescence, to the study of protein structure and dynamics has been extensively exploited to facilitate the understanding of complex biological problems. The interest in the application of phosphorescence, however, shows that new and complementary information can be had by careful optical studies of the phosphorescence lifetime. As in the early days of fluorescence spectroscopy in proteins, a complete and rigorous interpretation of the room temperature phosphorescence remains to be developed; nevertheless, it is clear that time-resolved phosphorescence yields new information on proteins in solution, for example, the detection of subtle conformational changes during protein folding, which is outside the sensitivity of earlier techniques. In addition, the great sensitivity of the phosphorescence lifetime to structural changes associated with rigidity and of nearby quenchers suggests that detailed structural information can be obtained when this approach is combined with the power of site-directed mutagenesis or other more biophysical techniques such as energy transfer to attached acceptors. We have presented basic aspects of time-resolved room temperature phosphorescence spectroscopy and demonstrated some useful features of the spectroscopic signals as well as the general approach to data analysis. However, it should be understood that extensions of this approach will easily allow faster and improved time resolution with greater sensitivity to highly quenched phosphorescing states. In addition, many extensions of this approach that are common to fluorescence spectroscopy have yet to be developed. For example, combining time-resolved phosphorescence with anaerobic stopped-flow techniques and more rapid data acquisition electronics will enable studies of conformational dynamics with considerably shortened dead times. Other possibilities include extending the preliminary studies of in vivo-based spectroscopy, such as to microscopy. In conclusion, time-resolved phosphorescence presents a new dimension to biophysical methodologies for the study of proteins, and it is likely that this area will continue to grow in capability as the fundamental understanding improves.

Animals↗
PubMedDate not supplied

Flexibility in proteins: tuning the sensitivity to O2 diffusion by varying the lifetime of a phosphorescent sensor in horseradish peroxidase.

The heme in horseradish peroxidase (HRP) was replaced by phosphorescent Pt-mesoporphyrin IX (PtMP), which acted as a phosphorescent marker of oxygen quenching and allowed comparison with another probe, Pd-mesoporphyrin IX (Khajehpour et al. (2003) Proteins 53, 656-666). Benzohydroxamic acid (BHA), a competitive inhibitor of the enzyme, was also used to monitor its effects on phosphorescence quenching. With the addition of BHA, in the presence of oxygen, the phosphorescence intensity of the protein increased. In contrast, the addition of BHA, in the absence of oxygen, reduced the phosphorescence intensity of the protein. K(d) = 18 microM when BHA binds to PtMP-HRP. The effect of BHA can be explained by two factors: (1) BHA reduces the accessibility of O(2) to the protein interior and (2) BHA itself quenches the phosphorescence. Consistent with this, the oxygen quenching of the phosphorescence of PtMP-HRP gave a quenching constant of k(q) = 234 mm Hg(-1) s(-1) in the absence of BHA and k(q) = 28.7 mm Hg(-1) s(-1) in the presence of BHA. The quenching rate of BHA is 4000 s(-1). The relative quantum yield of the phosphorescence of the Pt derivative is about six times that of the Pd derivative, whereas the phosphorescence lifetime is approximately eight times shorter. The high quantum yield and suitable lifetime make Pt-porphyrins appropriate as sensors of O(2) diffusion and flexibility in heme proteins.

Horseradish Peroxidase↗
PubMed1989

Characterization of lens alpha-crystallin tryptophan microenvironments by room temperature phosphorescence spectroscopy.

Room temperature phosphorescence techniques were used to study the structural and dynamic features of the tryptophan residues in bovine alpha-crystallin. Upon excitation at 290 nm, the characteristic signature of tryptophan phosphorescence was observed with an emission maximum at 442 +/- 2 nm. The phosphorescence intensity decay was biphasic with lifetimes of 5.4 ms (71%) and 42 ms (29%). Phosphorescence quenching measurements strongly suggest that each component corresponds to one class of tryptophans with the more buried residues having the longer emission lifetime. Three small-molecule quenchers were surveyed, and in order of increasing quenching efficiency: iodide less than nitrite less than acrylamide. A heavy-atom effect was observed in iodide solutions, and an upper limit of 5% was placed on the quantum yield of triplet formation in iodide-free solutions, while the phosphorescence quantum yield was estimated to be approximately 3.2 x 10(-4). The temperature dependence of the phosphorescence lifetime was measured between 5 and 40 degrees C. Arrhenius plots exhibited discontinuities at 26 and 29 degrees C for the short- and long-lived components, respectively, corresponding to abrupt transitions in segmental flexibility. Denaturation studies revealed conformational transitions between 1 and 2 M guanidine hydrochloride, and 4 and 6 M urea. Long-lived phosphorescence lifetimes of 3 and 7 ms were measured in 6 M guanidine hydrochloride and 8 M urea, respectively, suggesting that some structural features are preserved even at very high concentrations of denaturant. Our studies demonstrate the sensitivity of room temperature phosphorescence spectroscopy to the structure of alpha-crystallin, and the applicability of this technique for monitoring conformational changes in lens crystallin proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrylamide↗
PubMed1981

Optically detected magnetic resonance of the phosphorescent bases of Escherichia coli valine-specific transfer ribonucleic acid.

Phosphorescence spectroscopy and optical detection of triplet state magnetic resonance (ODMR) spectroscopy have been used to characterize bases that contribute to the phosphorescence emission of Escherichia coli valine-specific transfer ribonucleic acid. When it is excited with 335-nm light, a short-lived phosphorescence with an origin near 435 nm is observed and is assigned to 4-thiouridine (s4U) at position 8 of the tRNA sequence. With excitation at 290-300 nm, a structured, long-lived phosphorescence is observed with an origin near 380 nm, in addition to the s4U phosphorescence. Comparison was made of the phosphorescence and ODMR spectra between Mg2+-containing and Mg2+-free tRNA samples. The s4U phosphorescence of the Mg2+-containing sample is more structured, and the peak is blue shifted relative to the Mg2+-free sample. Both samples give a single low-frequency (ca. 2.9 GHz) ODMR signal, but the high-frequency signal region (ca. 19-20 GHz) is structured. The Mg2+-containing sample has a partially resolved group of lines centered at 19.3 GHz, whereas the Mg2+-free sample has two broad bands centered at 19.2 and 20.0 gHz. The differences are attributed to effects of Mg2+ on the tRNA conformation. The ODMR signals observed by monitoring the long-lived phosphorescence are assigned to a pyrimidine nucleoside, possibly 5-(carboxy-methoxy)uridine in the anticodon.

Base Sequence↗
PubMed2004

Quantitative determination of localized tissue oxygen concentration in vivo by two-photon excitation phosphorescence lifetime measurements.

This study describes the use of two-photon excitation phosphorescence lifetime measurements for quantitative oxygen determination in vivo. Doubling the excitation wavelength of Pd-porphyrin from visible light to the infrared allows for deeper tissue penetration and a more precise and confined selection of the excitation volume due to the nonlinear two-photon effect. By using a focused laser beam from a 1,064-nm Q-switched laser, providing 10-ns pulses of 10 mJ, albumin-bound Pd-porphyrin was effectively excited and oxygen-dependent decay of phosphorescence was observed. In vitro calibration of phosphorescence lifetime vs. oxygen tension was performed. The obtained calibration constants were kq = 356 Torr(-1) x s(-1) (quenching constant) and tau0 = 550 micros (lifetime at zero-oxygen conditions) at 37 degrees C. The phosphorescence intensity showed a squared dependency to the excitation intensity, typical for two-photon excitation. In vivo demonstration of two-photon excitation phosphorescence lifetime measurements is shown by step-wise PO2 measurements through the cortex of rat kidney. It is concluded that quantitative oxygen measurements can be made, both in vitro and in vivo, using two-photon excitation oxygen-dependent quenching of phosphorescence. The use of two-photon excitation has the potential to lead to new applications of the phosphorescence lifetime technique, e.g., noninvasive oxygen scanning in tissue at high spatial resolution. To our knowledge, this is the first report in which two-photon excitation is used in the setting of oxygen-dependent quenching of phosphorescence lifetime measurements.

Animals↗
PubMed1999

Phosphorescence of intermediates of the terminal stage of chlorophyll biosynthesis in plants

Phosphorescence (radiative triplet state deactivation) of the intermediates involved in chlorophyll a photobiosynthesis from protochlorophyllide (Pchld) was found in greening leaves at 77K. Photoactive Pchld forms showed phosphorescence with the major spectral bands at 920 and 970 nm and 2.5-3 msec lifetime. Photochemical formation of the primary non-fluorescent intermediates (NFI) was accompanied by quenching of Pchld fluorescence and phosphorescence, phosphorescence quenching being less efficient by a factor of two. The primary chlorophyllide form Chld 684/676 that appeared as a result of NFI dark transformation showed phosphorescence with a maximum at 980 nm and 2 msec lifetime. Secondary chlorophyllide forms Chld 690/680 and Chld 695/685, the products of photochemical Chld 684/676 transformation and intermediates in biosynthesis of chlorophyll of the light harvesting complex, emitted phosphorescence with maxima at 990-995 and 1005-1010 nm and 1-2 msec lifetimes. Chlorophyll Chl 675/668, the major product of the non-photochemical "side" reaction of Chld 684/676 that probably underlies the biogenesis of photosystem II, exhibited phosphorescence with a maximum at 954-960 nm and 2 msec lifetime. Analysis of the difference spectra of the side reaction products revealed a minor 930 nm phosphorescence band that might correspond to pheophytin a formed via the "side" reaction. Triplet states of the intermediates were not quenched by carotenoids, and the quantum yields of their population at 77K were not less than 10%. Under physiological conditions the triplet intermediates might be involved in photochemical reactions, in particular, in singlet oxygen photogeneration and photodestruction of the photosynthetic apparatus.

Journal Article↗
PubMed2003

Identification of the tautomeric form of formycin A in its complex with Escherichia coli purine nucleoside phosphorylase based on the effect of enzyme-ligand binding on fluorescence and phosphorescence.

Fluorescence and phosphorescence emission spectroscopy were employed to study the interaction of Escherichia coli purine nucleoside phosphorylase (PNP) with its specific inhibitor, formycin A (FA), a close structural analogue of adenosine (natural substrate), in the absence and presence of phosphate (P(i), substrate). Formation of enzyme-FA complexes led to marked quenching of enzyme tyrosine intrinsic fluorescence and phosphorescence, with concomitant increases in fluorescence and phosphorescence of FA. Fluorescence resonance energy transfer from the protein Tyr160 residue to the FA base moiety was identified as a major mechanism of protein fluorescence quenching, increased by addition of P(i). The effects of enzyme-FA interactions on the nucleoside excitation and emission spectra for fluorescence and phosphorescence revealed shifts in the tautomeric equilibrium of the bound FA, i.e. from the N(1)-H tautomer (predominant in solution) to the N(2)-H form, enhanced by the presence of P(i). The latter was confirmed by enzyme-ligand dissociation constant ( K(d)) values of 5.9+/-0.4 and 2.1+/-0.3 microM in the absence and presence of P(i), respectively. Addition of glycerol (80%, v/v) led to a lower enzyme affinity ( K(d) approximately 70 microM), without changes in binding stoichiometry. Enzyme-FA complex formation led to a higher increase of the fluorescence than the phosphorescence band of the ligand, consistent with the fact that the N(2)-H tautomer is characterized by a weaker phosphorescence than the N(1)-H tautomeric form. These results show, for the first time, the application of phosphorescence spectroscopy to the identification of the tautomeric form of the inhibitor bound by the enzyme.

Algorithms↗