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PubMed1993

Detection of intermediate protein conformations by room temperature tryptophan phosphorescence spectroscopy during denaturation of Escherichia coli alkaline phosphatase.

The reversible denaturation of Escherichia coli alkaline phosphatase (AP) was followed by monitoring changes in enzymatic activity as well as by measurements of the time-resolved room temperature phosphorescence from Trp 109. It is well known that the denaturants, ethylene diamine tetraacetic acid (EDTA), acid and guanidine hydrochloride (GdnHCl) inactive AP by different mechanisms as reflected by differences in the time dependence of inactivation. However, further information about structural changes that result during inactivation is obtained by measurement of the phosphorescence intensity and radiative decay rate. Time-resolved tryptophan phosphorescence is exquisitely sensitive to changes in the local environment of the emitting residue, unlike the steady state phosphorescence intensity which is a composite of both the lifetime and concentration of the emitting protein species. The results show that while inactivation in EDTA proceeds by loss of the zinc ion as expected, denaturation in acid or GdnHCl produces a heterogeneous population of AP molecules, detected by a distribution analysis of the phosphorescence lifetime, which may reflect multiple pathways to the final unfolded state. Time-resolved phosphorescence also demonstrates the existence of an enzymatically active but structurally less rigid intermediate state during unfolding. As the rigidity decreases, the susceptibility to further denaturation decreases at lower pH but increases with GdnHCl concentration. The experiments provide new insight into the mechanism of denaturation of AP and demonstrate the sensitivity of time-resolved room temperature phosphorescence to the structural details of intermediate states produced during unfolding of proteins.

Alkaline Phosphatase↗

PubMed1992

Phosphorescence and optically detected magnetic resonance measurements of the 2'AMP and 2'GMP complexes of a mutant ribonuclease T1 (Y45W) in solution: correlation with X-ray crystal structures.

Phosphorescence and ODMR measurements have been made on ribonuclease T1 (RNase T1), the mutated enzyme RNase T1 (Y45W), and their complexes with 2'GMP and 2'AMP. It is not possible to observe the phosphorescence of Trp45 in RNase T1 (Y45W). Only that of the naturally occurring Trp59 is seen. The binding of 2'GMP to wild-type RNase T1 produces only a minor red shift in the phosphorescence and no change in the ODMR spectrum of Trp59. However, a new tryptophan 0,0-band is found 8.2 nm to the red of the Trp59 0,0-band in the 2'GMP complex of the mutated RNase T1 (Y45W). Wavelength-selected ODMR measurements reveal that the red-shifted emission induced by 2'GMP binding, assigned to Trp45, occurs from a residue with significantly different zero-field splittings than those of Trp59, a buried residue subject to local polar interactions. The phosphorescence red shift and the zero-field splitting parameters demonstrate that Trp45 is located in a polarizable environment in the 2'GMP complex. In contrast with 2'GMP, binding of 2'AMP to RNase T1 (Y45W) induces no observable phosphorescence emission from Trp45, but leads only to a minor red shift in the phosphorescence origin of Trp59 in both the mutated and wild-type enzyme. The lack of resolved phosphorescence emission from Trp45 in RNase T1 (Y45W) implies that the emission of this residue is quenched in the uncomplexed enzyme. We conclude that local conformational changes that occur upon binding 2'GMP remove quenching residues from the vicinity of Trp45, restoring its luminescence.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Monophosphate↗

PubMed1983

Surface potential in rat liver mitochondria: terbium ion as a phosphorescent probe for surface potential.

The binding and phosphorescence of Tb3+ in rat liver mitochondria and submitochondrial particles were investigated. Mitochondria were treated briefly with N-ethyl-maleimide (NEM) to prevent phosphate leak and Tb3+ chelation. Up to 30 nmol of Tb3+/mg of protein binds to mitochondrial membranes with high apparent affinity (Kd congruent to 6 microM). Generation of a membrane potential had no significant effect on the apparent affinity or capacity of Tb3+ binding in NEM-treated mitochondria. Mitochondrial bound Tb3+ phosphorescence can be induced selectively by excitation of aromatic amino acid residues. The decay of mitochondrial bound Tb3+ phosphorescence is biphasic. The phosphorescence of the slow phase (t1/2 = 0.45-0.70 ms) is quenched by monovalent salts, indicating a negative surface potential at low salt medium of -5.4 +/- 2.8 mV [10 mM 3-(N-morpholino)-propanesulfonic acid, pH 7.2, 5 microM Tb3+]. In submitochondrial particles, a surface potential of -6.5 +/- 2.7 mV was estimated under the same conditions. Energization did not affect the surface potential significantly in submitochondrial particles and only slightly in mitochondria. Analysis of the phosphorescence of mitochondrial bound Tb3+ reveals two binding sites with high (Kd = 1.5 microM) and low affinity (Kd = 29 microM). The high-affinity site is tentatively identified as the Ca2+ carrier. A fraction of the carrier-bound Tb3+ phosphorescence decays rapidly, presumably as a result of energy transfer to cytochromes in the membrane core. These intramembrane sites appear to move to the surface on the generation of a membrane potential. We conclude that the salt effect on the phosphorescence of the slow phase may serve as a reliable measure of delocalized surface potential in mitochondria and submitochondrial particles. Tb3+ binding to the high-affinity site may be useful as a probe for the mitochondrial Ca2+ translocator.

Animals↗

PubMed1990

Phosphorescence and optically detected magnetic resonance studies of echinomycin-DNA complexes.

Echinomycin complexes with polymeric DNAs and model duplex oligonucleotides have been studied by low-temperature phosphorescence and optical detection of triplet-state magnetic resonance (ODMR) spectroscopy, with the quinoxaline chromophores of the drug used as intrinsic probes. Although not optically resolved, plots of ODMR transition frequencies versus monitored wavelength revealed heterogeneity in the phosphorescence emission of echinomycin, which was ascribed to the presence of two distinct quinoxaline triplet-state environments (referred to as the blue and red triplet states of echinomycin in this report). We think that a likely origin of the two triplet states of echinomycin is the occurrence of two or more distinct conformations of the drug in aqueous solutions. Spectroscopically observed perturbations of the triplet-state properties of echinomycin such as the phosphorescence emission spectrum, phosphorescence lifetime, ODMR spectrum, and zero-field splitting (zfs) energies were investigated upon drug binding to the double-stranded alternating copolymers poly(dG-dC).poly(dG-dC) [abbreviated as poly[d(G-C)2]] and poly(dA-dT).poly(dA-dT) [abbreviated as poly[d(A-T)2]], the homopolymer duplexes poly(dG).poly(dC) [abbreviated as poly(dG.dC)] and poly(dA).poly(dT) [abbreviated as poly(dA.dT)], and the natural DNAs from Escherichia coli, Micrococcus lysodeikticus, and calf thymus. Echinomycin bisintercalation complexes with the self-complementary oligonucleotides d(ACGT), d(CGTACG), and d(ACGTACGT), which are thought to model drug binding sites, were also investigated. Phosphorescence and ODMR spectroscopic results indicate that the quinoxaline chromophores of the drug are involved in aromatic stacking interactions in complexes with the natural DNAs as evidenced by red shifts in the phosphorescence 0,0 band of the drug, a small but significant reduction in the phosphorescence lifetime of the red triplet state, and reduction of the zfs D-value of both the blue and red triplet states upon drug complexation. These changes in the triplet-state properties of echinomycin are consistent with stacking interactions that increase the polarizability of the quinoxaline environment. The extent of the reduction of the D parameter for the red triplet state upon complexation with the polymeric DNAs was found to correlate with the binding affinities measured for these targets [Wakelin, L. P. G., & Waring, M. J. (1976) Biochem. J. 157, 721-740], with the single exception of the drug-poly[d(G-C)2] complex, for which an increase in the D-value was noted. In addition, upon drug binding to the natural DNAs, there is a reversal of signal polarity in the ODMR spectra of the red triplet state. Among the synthetic DNA polymers investigated, a reversal of ODMR signal polarity was found only with the echinomycin-poly[d(A-T)2] complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗

PubMed1992

Time-resolved circularly polarized protein phosphorescence.

The existence of circular polarization in room-temperature protein phosphorescence is demonstrated, and time-resolved circularly polarized phosphorescence (TR-CPP) is used to characterize unique tryptophan environments in multitryptophan proteins. Circularly polarized luminescence studies provide information regarding the excited state chirality of a lumiphore which can be used to extract sensitive structural information. It is shown by time resolving the circular polarization that it is possible to correlate the excited state chirality with unique decay components in a multiexponential phosphorescence decay profile. The present study presents a concurrent analysis of room-temperature time-resolved phosphorescence and TR-CPP of bacterial glucose-6-phosphate dehydrogenase as well as those of horse liver alcohol dehydrogenase. Only one of the two tryptophan residues per subunit of dimeric alcohol dehydrogenase is believed to phosphorescence, while the dimeric glucose-6-phosphate dehydrogenase has eight tryptophan residues per subunit and shows a corresponding complexity in its phosphorescence decay profile. The anisotropy factor [g(em) = delta I/(Itotal/2); delta I = Ileft circular-Iright circular] for alcohol dehydrogenase is time independent, suggesting a unique excited state chirality. The phosphorescence decay of glucose-6-phosphate dehydrogenase can be well fitted with four exponential terms of 4, 23, 76, and 142 msec, and the TR-CPP of this enzyme shows a strong time dependence that can be resolved into four individual time-independent anisotropy factors of -4.0, -2.1, +6.5, and +6.9 (x10(-3)), each respectively associated with one of the four lifetime components. These results demonstrate how the use of TR-CPP can facilitate the study of proteins with multiple lumiphores.

Alcohol Dehydrogenase↗

PubMed2004

Measurement of muscle microvascular oxygen pressures: compartmentalization of phosphorescent probe.

OBJECTIVE: To determine whether the phosphorescent probe Oxyphor R2 (a palladium porphyrin dendrimer) becomes extravasated within normotensive skeletal muscle, R2 perfusion and washout studies were performed using a perfused rat hindlimb preparation. METHODS: Phosphorescence signals were monitored in tibialis anterior muscles after 35 min of R2 blood perfusion and across a subsequent washout period that included vasodilation (sodium nitroprusside, SNP, approximately 3 x 10(-2) M). RESULTS: Two responses were evident: Group 1 (n = 4)--Inflowing blood pressure and vascular conductance remained stable close to initial values and subsequently a marked vasodilation was evident with SNP (vascular conductance; R2 blood perfusion, 0.096 +/- 0.005; washout, pre-SNP, 0.085 +/- 0.005, post-SNP, 0.110 +/- 0.005 mL/min/mmHg, p <.05, for pre- vs. post-SNP). Baseline phosphorescence signals could be monitored up to 99 +/- 36 s post-SNP when the phosphorescence signal disappeared. For these muscles, palladium content was undetectable. Group 2 (n = 3)--Inflowing blood pressure increased 112% and vascular conductance fell approximately 50%. These hindlimbs were unresponsive to SNP, phosphorescence signal was undiminished by washout and SNP, and muscles became edematous. CONCLUSIONS: These results suggest that in normotensive muscle (i.e., Group 1 above), extravasation of phosphorescent probe R2 over 35 min of perfusion is insufficient to yield a detectable phosphorescence signal in skeletal muscle.

Animals↗

PubMed1999

Phosphorescence quenching method for measurement of intracellular PO2 in isolated skeletal muscle fibers.

Values of skeletal muscle intracellular PO2 during conditions ranging from rest to maximal metabolic rates have been difficult to quantify. A method for measurement of intracellular PO2 in isolated single skeletal muscle fibers by using O2-dependent quenching of a phosphorescent-O2 probe is described. Intact single skeletal muscle fibers from Xenopus laevis were dissected from the lumbrical muscle and mounted in a glass chamber containing Ringer solution at 20 degreesC. The chamber was placed on the stage of an inverted microscope configured for epi-illumination. A solution containing palladium-meso-tetra (4-carboxyphenyl) porphine bound to bovine serum albumin was injected into single fibers by micropipette pressure injection. Phosphorescence-decay curves (average of 10 rapid flashes) were recorded every 7 s from single cells (n = 24) in which respiration had been eliminated with NaCN, while the PO2 of the Ringer solution surrounding the cell was varied from 0 to 159 Torr. For each measurement, the phosphorescence lifetime was calculated at the varied extracellular PO2 by obtaining a best-fit estimate by using a monoexponential function. The phosphorescence lifetime varied from 40 to 70 microseconds at an extracellular PO2 of 159 Torr to 650-700 microseconds at 0 Torr. The phosphorescent lifetimes for the varied PO2 were used to calculate, by using the Stern-Volmer relationship, the phosphorescence-quenching constant (100 Torr-1. s-1), and the phosphorescence lifetime in a zero-O2 environment (690 microseconds) for the phosphor within the intracellular environment. This technique demonstrates a novel method for determining intracellular PO2 in isolated single skeletal muscle fibers.

Animals↗

PubMed2004

Characterization of f-actin tryptophan phosphorescence in the presence and absence of tryptophan-free myosin motor domain.

The effect of binding the Trp-free motor domain mutant of Dictyostelium discoideum, rabbit skeletal muscle myosin S1, and tropomyosin on the dynamics and conformation of actin filaments was characterized by an analysis of steady-state tryptophan phosphorescence spectra and phosphorescence decay kinetics over a temperature range of 140-293 K. The binding of the Trp-free motor domain mutant of D. discoideum to actin caused red shifts in the phosphorescence spectrum of two internal Trp residues of actin and affected the intrinsic lifetime of each emitter, decreasing by roughly twofold the short phosphorescence lifetime components (tau(1) and tau(2)) and increasing by approximately 20% the longest component (tau(3)). The alteration of actin phosphorescence by the motor protein suggests that i), structural changes occur deep down in the core of actin and that ii), subtle changes in conformation appear also on the surface but in regions distant from the motor domain binding site. When actin formed complexes with skeletal S1, an extra phosphorescence lifetime component appeared (tau(4), twice as long as tau(3)) in the phosphorescence decay that is absent in the isolated proteins. The lack of this extra component in the analogous actin-Trp-free motor domain mutant of D. discoideum complex suggests that it should be assigned to Trps in S1 that in the complex attain a more compact local structure. Our data indicated that the binding of tropomyosin to actin filaments had no effect on the structure or flexibility of actin observable by this technique.

Actins↗

PubMed1999

Time resolved fluorescence and phosphorescence properties of the individual tryptophan residues of barnase: evidence for protein-protein interactions.

Steady-state and time-resolved fluorescence, as well as phosphorescence measurements, were used to resolve the luminescence properties of the three individual tryptophan residues of barnase. Assignment of the fluorescence properties was performed using single-tryptophan-containing mutants and the results were compared with the information available from the study of wild-type and two-tryptophan-containing mutants (Willaert, Lowenthal, Sancho, Froeyen, Fersht, Engelborghs, Biochemistry 1992;31:711-716). The fluorescence and the phosphorescence emission of wild-type barnase is dominated by Trp35, although Trp71 has the strongest intrinsic fluorescence when present alone. Fluorescence emission of these two tryptophan residues is blue-shifted and pH-independent. The fluorescence decay parameters of Trp94 are pH-dependent, and an intramolecular collision frequency of 2 to 5 x 10(9) s(-1) between Trp94 and His18 is calculated. Fluorescence emission of Trp94 is red-shifted. Fluorescence anisotropy decay reveals the local mobility of the individual tryptophan residues and this result correlates well with their phosphorescence properties. Trp35 and Trp71 display a single phosphorescence lifetime, which reflects the rigidity of their environment. Surface Trp94 does not exhibit detectable phosphorescence emission. The existence of energy transfer between Trp71 and Trp94, as previously detected by fluorescence measurements, is also observed in the phosphorescence emission of barnase. Dynamic quenching causes the phosphorescence intensity to be protein-concentration dependent. In addition, fluorescence anisotropy shows concentration dependency, and this can be described by the formation of trimers in solution.

Bacterial Proteins↗

PubMed1992

Localization of tumors and evaluation of their state of oxygenation by phosphorescence imaging.

Oxygen-dependent quenching of phosphorescence has been used to image the distribution of oxygen pressure in small tumors and surrounding tissue. Suspensions of cultured 9L cells or small pieces of solid tumors from 9L cells were injected into the surface of the muscle of the hindquarter of rats, and the tumors were grown until they were 0.2-1.0 cm in diameter. The phosphorescent probe for oxygen was injected into the systemic blood, and phosphorescence was imaged with a video camera. Images of the phosphorescence were collected using a series of different delay times after illumination with a light flash (less than 5-microseconds width at half-height), and the phosphorescence decay constants (lifetimes) and oxygen pressure were calculated for each pixel of the image arrays. The areas of tissue within the tumors were observed to have increased phosphorescence lifetimes and lower oxygen pressures than the surrounding tissue. Phosphorescence imaging is, therefore, a noninvasive optical method which permits quantitation of the distribution of oxygen in small tumors and also, at least in the 9L tumors, differentiation of tumor from normal tissue.

Animals↗

PubMedDate not supplied

[Chlorophyll phosphorescence in leaves and cells of algae].

Phosphorescence and delayed fluorescence of chlorophyll have been observed in frozen suspensions of green, blue-green and red alga, normal and mutant leaves of pea, kidney bean and maize at the temperature range from --196 to --70 degrees C. The phosphorescence maximum lies at 960--980 nm, excitation maximum--at 674+/-2 nm, lifetime is 1.5--2.5 msec, quantum yield vaires in the range of 0.03--3 x 10(-6), being much lower than that of chlorophyll phosphorescence in solutions. The weakest phosphorescence was found in normal leaves, the strongest one--in cells of Scendesmus and leaves of xi-carotene mutants of maize. The data suggest that phosphorescence belongs to antenna chlorophyll and phosphorescence quenching is due to energy migration from singlet and triplet states of antenna to non-phosphorescent forms of chlorophyll and carotenoids.

Chlorophyll↗

PubMed2003

Laser-induced quenched phosphorescence detection in capillary electrophoresis.

The feasibility of laser-based excitation for quenched phosphorescence detection in capillary electrophoresis (CE) was explored for the first time by using a small-size, quadrupled Nd-YAG laser emitting 266 nm pulses (duration, 0.4 ns) at a repetition rate of 7.8 kHz. To provide a continuous phosphorescence background, the phosphorophore 1-bromo-4-naphthalene sulfonic acid (BrNS) was added to the separation buffer. Both experiments and theory show that in laser-induced phosphorescence (LIP) - in contrast with lamp-excited phosphorescence - one normally deals with such high triplet-state phosphorophore concentrations that triplet-triplet annihilation is the major deactivation pathway. This results in a lower quantum yield of the analyte-induced bimolecular quenching interaction and, thus, the observed quenching signal. The situation can be improved by using a cylindrical lens for excitation in order to reduce the irradiance. In this case limits of detection (LODs) similar to those obtained using lamp excitation (1x10(-8) M) were achieved, while the width of the detection window was reduced from about 4 mm to 1 mm. Even under exclusion of triplet-triplet annihilation, i.e., under conditions of low irradiance, for our setup the quenching yields in LIP were smaller than in lamp-based phosphorescence detection. This is due to the repetition rate of the laser (7.8 kHz), which is too high in view of the phosphorescence lifetime (ca. 300 micros at low irradiance). Theory shows that this disadvantageous effect will be fully eliminated if the repetition rate is decreased to 1 kHz.

Electrons↗

PubMed1998

Phosphorescence emission of 7-azatryptophan and 5-hydroxytryptophan in fluid solutions and in alpha2 RNA polymerase.

The tryptophan analogues 7-azaindole (7-Aza W) and 5-hydroxytryptophan (5-OH W) have a significant absorbance between 310-320 nm, which allows them to act as selective luminescence probes in protein mixtures containing a large number of tryptophan residues. To assess the potential of their phosphorescence emission in probing the nature of protein environments the delayed emission was examined as a function of temperature and solvent viscosity. Whereas in low temperature (135 K) propylene glycol/buffer glasses the phosphorescence of both 7-aza W and 5-OH W is structured, intense and exhibit a lifetime of a few seconds, above the glass transition temperature (180 K) the delayed emission is considerably quenched. Temperature profiles show that as the solvent is more fluid the phosphorescence of 5-OH W becomes red shifted, poorly structured and the triplet lifetime drops steeply reaching 29 micro(s) in buffer at 274 K. For 7-aza W the alterations are more drastic and no phosphorescence could be detected above 193 K. This implies that in fluid aqueous media the excited triplet state of these analogues is deactivated by vary efficient nonradiative processes. The quenching of 5-OH phosphorescence is not prevented even when the chromophore is inserted in a solvent protected protein environment. Indeed, substitution of the single Trp of a2 RNA Polymerase, which according to its relatively low phosphorescence lifetime at ambient temperature is substantially shielded from the solvent, with 5-OH did not inhibit the quenchability of the latter. Knowledge of the quenching mechanisms is therefore needed for this emission to report on the nature of the protein environment.

5-Hydroxytryptophan↗

PubMed1992

Monitoring of the oxygen pressure in the blood of live animals using the oxygen dependent quenching of phosphorescence.

Oxygen dependent quenching of phosphorescence is a powerful new method for measuring oxygen pressure in biological systems (see Vanderkooi et al, J. Biol. Chem. 262 (1987) 5476; Wilson et al, J. Biol. Chem. 263 (1988) 2712). This technology has now been extended to include measurements of the phosphorescent of oxygen probes dissolved in the blood as a complex with albumin. In this communication, we report on a new microcomputer controlled phosphorimeter in which the tissue is illuminated by a flash lamp and the phosphorescence observed through flexible light guides designed to make measurements in regions down to approximately 1 mm in diameter. Measurements of the phosphorescence lifetimes of these probes in regions of tissue necessarily report a distribution of oxygen pressures due to the presence of blood in veins and arteriols as well as that present in the capillaries. Thus the phosphorescence decay is the sum of a continuum of exponentials with different decay constants and initial intensities. The complete phosphorescence decay curve is digitized using a 12 bit, 1 MHz A/D board and deconvoluted using numerical methods to yield a distribution of exponentials contributing to the total decay curve.

Animals↗

PubMed1992

Oxygen dependent quenching of phosphorescence: a perspective.

Oxygen quenches phosphorescence by energy transfer from the phosphor when oxygen molecules collide with molecules of the phosphor in the excited triplet state. Thus increasing oxygen pressure causes an increase in the rate of decay of phosphorescence (shorter lifetimes) and a decrease in total phosphorescence intensity. Phosphors have been selected which decay with a single exponential and for which the relationship between phosphorescence lifetime and oxygen pressure is quantitatively described by the Stern-Volmer equation. The use of phosphorescence lifetime as the measure of oxygen pressure makes the method insensitive to the absorbance changes of other chromophores in the system. This method has permitted quantitative, rapid (less than 10 msec) and sensitive (to less than 10(-8) Torr) measurements of oxygen pressure in suspensions of cells or subcellular organelles. In tissues, oxygen pressure has been evaluated by measuring phosphorescence using an intensified CCD camera. Maps of oxygen pressure in the vasculature of the cortex of the brain and of other tissues demonstrate the method is limited only by the optics of the system and resolutions of a few microns are readily attained.

Animals↗

PubMed2006

The influence of human serum albumin on the photogeneration of singlet oxygen by meso-tetra(4-sulfonatophenyl)porphyrin. An infrared phosphorescence study.

meso-Tetra(4-sulfonatophenyl)porphyrin (TPPS4) is a water soluble photosensitizer, which is currently clinically tested as a PDT drug. In our contribution, we present IR spectral- and time-resolved phosphorescence data reflecting the influence of human serum albumin (HSA) on singlet oxygen photogeneration by TPPS4. IR emission of TPPS4 was studied in samples containing various concentrations of HSA in phosphate buffer. The observed changes in spectral and temporal behaviour of TPPS4 and singlet oxygen phosphorescence caused by the addition of HSA are equivalent to the effect of nitrogen purging of HSA-free solutions of TPPS4. The main feature induced by addition of HSA appears to be the occurrence of a long-lived (tens of microseconds) photosensitizer phosphorescence at 900 nm besides ordinary short-lived (approximately 2 micros) one at 820 nm. It is accompanied by presence of a long-lived component of singlet oxygen emission with lifetime roughly corresponding to that of the long photosensitizer phosphorescence component. Moreover, the quantum yield of singlet oxygen phosphorescence decreases with increasing HSA concentration, while total quantum yield of TPPS4 phosphorescence rises. These facts are explained by a shielding effect of HSA on bound molecules of TPPS4 against quenching by oxygen which is analogous to oxygen removal by nitrogen purging.

Buffers↗

PubMed1981

Phosphorescence of alkaline phosphatase of E. coli in vitro and in situ.

Escherichia coli K-12, which is rich in alkaline phosphatase, exhibits phosphorescence characteristic of tryptophan at room temperature. E coli mutants which do not have alkaline phosphatase do not show long-lived phosphorescence. The phosphorescence spectrum and lifetime of E. coli K-12 was similar to that of purified alkaline phosphatase from E. coli. These results indicate that the long-lived tryptophan phosphorescence in E. coli is likely to be derived from alkaline phosphatase in situ. The temperature dependence of tryptophan phosphorescence life-time of purified alkaline phosphatase and E. coli K-12 differ; this may imply that alkaline phosphatase in E. coli may be associated with the cell envelope and is therefore protected against structural changes in the protein which result in increased phosphorescence decay rates.

Alkaline Phosphatase↗

PubMed2005

Determination of trace copper by solid substrate-room temperature phosphorescence quenching method based on activating effect of alpha,alpha'-dipyridyl on Vitamin C reducing beryllon.

A new solid substrate-room temperature phosphorescence quenching method for the determination of trace copper has been established. It is based on the fact that beryllon (R) can emit strong and stable solid substrate-room temperature phosphorescence on the filter paper, and Vitamin C (Vc) reduces R to non-phosphorescent compound that leads to solid substrate-room temperature phosphorescence (SS-RTP) quenching of R, and alpha,alpha'-dipyridyl can activate copper catalyzing Vitamin C reducing R. The DeltaI(p) of the system with alpha,alpha'-dipyridyl is 3.3 times higher than that without alpha,alpha'-dipyridyl, which shows the reaction of alpha,alpha'-dipyridyl activating copper catalyzing Vitamin C reducing R. The reducing value of phosphorescence intensity (DeltaI(p)) is directly proportional to the content of Cu(II) in the range of 0.040-4.0 fg spot(-1) (corresponding concentration: 0.10-10.0 pg ml(-1), sample volume: 0.40 microlspot(-1)). The regression equation of working curve can be expressed as DeltaI(p)=69.99+41.00 m Cu(2+) (fg spot(-1)) (r=0.9980, n=6), and the detection limit is 0.0088 fg spot(-1)(corresponding concentration: 2.2 x 10(-14) g ml(-1)). This sensitive and accurate method with good repeatability and high selectivity has been applied to the determination of trace copper in real samples with satisfactory results. The reaction mechanism for the determination of trace copper by solid substrate-room temperature phosphorescence quenching method based on the activating effect of alpha,alpha'-dipyridyl on Vitamin C reducing beryllon is also discussed.

Ascorbic Acid↗