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PubMed1997

Pressure-induced dissociation of yeast glyceraldehyde-3-phosphate dehydrogenase: heterogeneous kinetics and perturbations of subunit structure.

In studies of pressure-induced subunit dissociation of oligomeric proteins, the thermodynamic dissociation constant and the dissociation volume change are derived by assuming that high pressure itself does not significantly perturb the structure of both oligomer and isolated subunit. In this report, the intrinsic phosphorescence emission of Trp reveals that high-pressure dissociation of tetrameric yeast glyceraldehyde-3-phosphate dehydrogenase results in a dramatic shortening of the phosphorescence lifetime, from 300 to less than 2 ms, that is consistent with a profound loosening of the polypeptide structure about the phosphorescence probe. On pressure release, subunit reassociation occurs readily whereas recovery of the native phosphorescence properties is a very slow, thermally activated, process which goes hand in hand with the recovery of the catalytic activity. Further, the comparison between the kinetic traces that describe the degree of dissociation and the change in phosphorescence lifetime, at various applied pressures, has established the following: (1) that high pressure plays a direct role on the structural rearrangement, the extent of which increases with pressure; (2) that the conformational change in the monomer is concomitant with, or follows closely after, the break up of the tetramer, in any case long before an apparent tetramer-monomer equilibrium is established; (3) that native tetramers are highly heterogeneous with regard to their rate of dissociation. The influence of temperature, of protein concentration, of binding of NAD+, and of the addition of 2 M urea on the dissociation/phosphorescence kinetic profiles was also examined. The complications arising from these conformational changes for the derivation of the dissociation free energy change as well as their relevance for understanding the lack of concentration dependence of the degree of dissociation are discussed.

Adenosine Triphosphate↗

PubMed2003

Red blood cell velocity and oxygen tension measurement in cerebral microvessels by double-wavelength photoexcitation.

Because the regulation of microcirculation in the cerebral cortex cannot be analyzed without measuring the blood flow dynamics and oxygen concentration in cerebral microvessels, we developed a fluorescence and phosphorescence system for estimating red blood cell velocity and oxygen tension in cerebral microcirculation noninvasively and continuously with high spatial resolution. Using red blood cells labeled with fluorescent isothiocyanate to visualize red cell distribution and using the oxygen quenching of Pd-meso-tetra-(4-carboxyphenyl)-porphyrin phosphorescence to measure oxygen tension enabled simultaneous measurement of blood velocity and oxygen tension. We examined how the measurement accuracy was affected by the spatial resolution and by the excitation laser light passing through the targeted microvessel and exciting the oxygen probe dye in the tissue beneath it. Focusing the excitation light into the microvessel stabilized the phosphorescence lifetime at each spatial resolution; moreover, it greatly reduced phosphorescence from the brain tissue. Animal experiments involving acute hemorrhagic shock demonstrated the feasibility of our system by showing that the changes in venular velocity and oxygen tension are synchronized to the change in mean arterial pressure. Our system measures the red cell velocity and oxygen concentration in the cerebral microcirculation by using the differences in luminescence and wavelength between fluorescence and phosphorescence, making it possible to easily acquire information about cerebral microcirculatory distribution and oxygen tension simultaneously.

Animals↗

PubMed2002

[Applications of phosphorimetry in pharmaceutical analysis].

Applications of phosphorimetry including solid substrate phosphorescence, liquid medium phosphorescence, low temperature phosphorescence and phosphorescence sensors were reviewed in pharmaceutical analysis. The drugs involved here included the varieties of alkaloid, Chinese traditional medicine, tetracyclines, quinolone, riboflavin, anticancer medicine, naphazoline, naproxen, nafronyl dipyridamole and so on. Solid surface phosphorimetry is characterized by sample volume of microliter grade, simple and fast operation procedures in pharmaceutical analysis. The combination of liquid phosphorescence with flow injection analysis and chemosensing technique has good advantages in fast, continuous and on-line monitoring of medicines. Modified low temperature phosphorimetry still remains its high sensitivity and overcomes some disadvantages in the procedures. Phosphorimetry will be more widely applied to pharmaceutical analysis as the development of sensitive and quenching, energy transfer, derivative and immunization luminescence.

Alkaloids↗

PubMedDate not supplied

Feasibility of noninvasive imaging of chorioretinal oxygenation.

BACKGROUND AND OBJECTIVE: The feasibility of an optical system for noninvasive imaging of chorioretinal oxygenation was evaluated. Due to its depth discrimination, this optical section imaging technique has potential for differential imaging of oxygen tension in the chorioretinal vasculatures. MATERIALS AND METHODS: The method consisted of projecting a narrow laser line obliquely on the retina after intravenous injection of an oxygen-sensitive probe and imaging the phosphorescence emission. Due to the angle between the incident laser and imaging path, a phosphorescence optical section image of the retina was captured. The phosphorescence intensity was measured in the chorioretinal vasculatures. The method was tested in three rats while breathing 10% oxygen, 50% oxygen, and room air. RESULTS: On the phosphorescence optical section image, vasculatures appeared laterally displaced according to their depth location, displaying probe phosphorescence separately in the chorioretinal vasculatures. Oxygenation increased in all vasculatures with increased inhaled percent oxygen. Oxygenation in the retinal artery was 2.3, 1.9, and 1.6 times oxygenation in the retinal vein, capillary, and choroid, respectively. During hypoxia, oxygenation decreased by 28%, 18%, 22%, and 14% in the retinal vein, artery, capillary, and choroid, respectively. During hyperoxia, oxygenation increased by 30%, 45%, 36%, and 28% in the retinal vein, artery, capillary, and choroid, respectively. CONCLUSION: The results demonstrate the feasibility of this technique for noninvasive and separate imaging of chorioretinal oxygenation and its potential for three-dimensional oxygen tension imaging.

Animals↗

PubMedDate not supplied

[Analytical description of electron-vibrational protein spectra].

Electron-vibrational spectra of phosphorescence and fluorescence of tryptophan residues in proteins at 77 K are best approximated by theoretical curves computed according to a model which suggests the existence of two independent series of Gaussian vibrational components. Each series contains one type of vibrations. Phosphorescence and fluorescence spectra of proteins with various localizations of their single tryptophan residue were fitted by a curve computed according to this model. The results obtained show that the phosphorescence band of tryptophan residues in proteins seems to contain two types of vibrations with frequencies 650-800 cm-1 and 1350-1500 cm-1. Since the substitution of H2O by D2O does not change the frequencies of both vibrations in the phosphorescence spectra of human serum albumin, melittin and tryptophan in 1 M KCl, it is reasonable to suggest that the 1350-1500 cm-1 series corresponds to the W5 type vibrations (B19a type of vibrations of benzene ring). The 650-800 cm-1 series"can be identified with W18 type of vibrations (breathing vibrations of indole ring). Phosphorescence parameters of tryptophan residues in proteins correlate with their fluorescence parameters.

Animals↗

PubMed1999

Spectral properties of Trp182, Trp194, and Trp250 on the alpha subunit of bacterial luciferase.

The phosphorescence and fluorescence properties of bacterial luciferase (alphabeta) mutants from Xenorhabdus luminescens were investigated. All tryptophans in the alpha and beta subunits were replaced with tyrosines except for one or two tryptophans in the alpha subunit. Because one luciferase mutant (W250) retained only a single tryptophan in the alpha subunit while two other mutants (W182/250 and W194/250) each contained two tryptophans in the alpha subunit, it was possible to deduce the spectral properties of these specific tryptophans (Trp182, Trp194, Trp250). Analyses of the phosphorescence properties were particularly revealing as only a single phosphorescence emission peak at 411-414 nm was observed for the W250 and W194/250 mutants while peaks at 409 and 414 nm could be clearly observed for the W182/250 mutant. Coupled with intrinsic fluorescence quenching experiments, these results show that alphaTrp182 is in a distinctly polar environment while alphaTrp250 is in a hydrophobic region and illustrate the advantages of using phosphorescence to recognize different microenvironments for tryptophan residues.

Amino Acid Substitution↗

PubMed1996

Pressure effects on the structure of oligomeric proteins prior to subunit dissociation.

In studies of pressure-induced subunit dissociation of protein aggregates, now widely used to evaluate the association free energy, entropy and enthalpy of very stable complexes, it is assumed that high pressure does not influence their structure/thermodynamic parameters and that some peculiarities of these equilibria, such as the decrease in subunit affinity at larger degrees of dissociation (alpha) and hysteresis in alpha/pressure diagrams are imputable to the slow conformational drift of isolated subunits. To test this premise, the conformation of dimeric alcohol dehydrogenase from horse liver and alkaline phosphatase from Escherichia coli was monitored as a function of pressure (up to 3 kbar) and temperature (0 to 50 degrees C) by means of the intrinsic Trp fluorescence and phosphorescence emission and binding of the 1-anilinonaphatalene-8-sulphonic acid (ANS) fluorophore. The results show a distinct influence of high pressure on the native dimers whose changes in conformation may, depending on whether or not these alterations are promptly reversed, be distinguished in elastic and inelastic changes. Elastic changes are ubiquitous and refer to pronounced modulations of the phosphorescence lifetime which is a monitor of the internal flexibility of the macromolecules. They attest to a tightening of the globular structure in the lower pressure range (below 1.5 kbar) as opposed to an increased fluidity in the higher range. The trend is similar between the two proteins and the tightening/loosening effect is fully consistent with the role that internal cavities and hydration of polypeptide is expected to play in determining the compressibility of these biopolymers. Inelastic perturbations reveal a more profound loosening of the globular fold and were observed only with alcohol dehydrogenase under conditions (low temperature (t < 10 degrees C) and high pressure (p > 2.5 kbar)) that favour protein hydration. They involve slow consecutive reactions that produce drastic reductions in phosphorescence lifetime, spectral red shifts, quenching of fluorescence and phosphorescence emission and modulation of ANS binding. Judging from the full protection afforded by glycerol as cosolvent, or the remarkable enhancement caused by modest concentrations of urea, the driving force of these perturbations appears to be pressure-induced hydration of the protein. Inelastic conformational changes are accompanied by a slow and often incomplete recovery of enzymatic activity. The characteristic times of these processes, their pressure dependence and the slow, thermally activated, reversibility are discussed in the light of hysteresis phenomena and changes of subunit affinity in dissociation equilibria.

Alcohol Dehydrogenase↗

PubMed1997

Oxygen distribution in the vasculature of mouse tissue in vivo measured using a near infra red phosphor.

Oxygen dependent quenching of phosphorescence has been used to measure the oxygenation of tissue in mice, including the differences between normal tissue and that of a murine tumor. Approximately 0.3 mg of the phosphorescence oxygen probe, Green 2W, was injected into the tail vein of tumor bearing mice. The mice were immobilized using an anesthetic cocktail and illuminated with flashes (< 4 microseconds t1/2) of light of 636 +/- 15 nm. The emitted phosphorescence (790 nm max.) was measured using an imaging phosphorimeter with an intensified CCD camera, an instrument which provides two dimensional digital maps of oxygen pressure. Both the illumination light and the phosphorescence were in the near infra red region of the spectrum, where skin and tissue have little absorption. The light can therefore readily pass through the skin and centimeter thickness of tissue. Mice are sufficiently small that the oxygen pressure maps could be obtained by illuminating from either the same or the opposite side as the camera (and tumor). The tumors were observed as regions with oxygen pressures substantially below those of the surrounding normal tissue. Thus, it is possible to non-invasively detect these tumors and to monitor their internal oxygen pressure in real time and through cm of tissue.

Animals↗

PubMed2003

Sensitive and simple determination of the vasodilator agent dipyridamole in pharmaceutical preparations by phosphorimetry.

The applicability of heavy atom-induced room-temperature phosphorescence to pharmaceutical samples is demonstrated in this work. Thus a new, simple, rapid, and selective phosphorimetric method for dipyridamole determination is proposed. The phosphorescence signals are a consequence of intermolecular protection when analytes are exclusively in the presence of heavy atom salts and sodium sulfite as an oxygen scavenger to minimize RTP quenching. The determination was performed in 0.1 mol L(-1) thallium(I) nitrate and 8 mmol L(-1) sodium sulfite at a measurement temperature of 20 degrees C. The phosphorescence intensity was measured at 635 nm, with excitation at 305 nm. Phosphorescence was easily developed; a linear concentration range was obtained between 0 and 100 ng mL(-1) with a detection limit of 940 ng L(-1), an analytical sensitivity of 2.5 ng mL(-1), and a standard deviation of 2.7% at 60 ng mL(-1) concentration. The method has been successfully applied to the analysis of dipyridamole in a unique Spanish commercial formulation containing 100 ng mL(-1) per capsule. The recovery was 101.6% with 6.5% standard deviation of analytical measurement. The method using the standard addition methodology has been validated.

Calibration↗

PubMed1988

Phosphorimeters for analysis of decay profiles and real time monitoring of exponential decay and oxygen concentrations.

A phosphorimeter which can be assembled at low cost from mainly commercially available components and which has better time resolution, data acquisition rate, sensitivity, and flexibility than commercially available instruments is described. As a phosphorescence analyzer the instrument can measure phosphorescence lifetimes ranging from approximately 30 microseconds to seconds from samples with variable intensity, excitation, and emission spectra and which may follow complex decay behavior. Configured as a phosphorescence monitor it is designed for fast, repetitive calculation of phosphorescence lifetime, assuming single-exponential decay, and can be used to calculate oxygen concentration in biological samples in real time.

Luminescent Measurements↗

PubMed1984

Optically detected zero field magnetic resonance characterization of a bromine atom-containing polynucleotide, poly(dA-br5dU).

Phosphorescence and optically detected zero field magnetic resonance ( ODMR ) spectra are reported for a bromine atom-containing polynucleotide, poly(dA- br5dU ). The triplet state luminescence of poly(dA- br5dU ) is dominated by the phosphorescence of the bromouracil base which possesses sub-millisecond triplet lifetimes. Characteristic multiple slow passage ODMR transitions, which are observed in both br5dUrd and poly(dA- br5dU ), are assigned to the triplet state of bromouracil. In addition, an abnormally-perturbed adenine triplet state, which is not apparent in the phosphorescence spectrum of poly(dA- br5dU ), is detected and identified by its slow passage ODMR and amplitude-modulated phosphorescence microwave double resonance spectra. It is proposed that the perturbed adenine is a minor component of the polynucleotide structure which is present in regions of altered stacking induced by the high polarizability of the Br atom.

Adenine↗

PubMed2004

Tryptophan interactions with glycerol/water and trehalose/sucrose cryosolvents: infrared and fluorescence spectroscopy and ab initio calculations.

In order to correlate how the solvent affects emission properties of tryptophan, the fluorescence and phosphorescence emission spectra of tryptophan and indole model compounds were compared for solid sugar glass (trehalose/sucrose) matrix and glycerol/water solution and under the same conditions, these matrices were examined by infrared spectroscopy. Temperature was varied from 290 to 12 K. In sugar glass, the fluorescence and phosphorescence emission spectra are constant over this temperature range and the fluorescence remains red shifted; these results are consistent with the static interaction of OH groups with tryptophan in the sugar glass. In sugar glass containing water, the water retains mobility over the entire temperature range as indicated by the HOH infrared bending frequency. The fluorescence of tryptophan in glycerol/water shifts to the blue as temperature decreases and the frequency change of the absorption of the HOH bend mode is larger than in the sugar glass. These results suggest rearrangement of glycerol and water molecules over the entire temperature change. Shifts in the fluorescence emission maximum of indole and tryptophan were relatively larger than shifts for the phosphorescence emission-as expected for the relatively smaller excited triplet state dipole for tryptophan. The fluorescence emission of tryptophan in glycerol/water at low temperature has maxima at 312, 313, and 316 nm at pH 1.4, 7.0, and 10.6, respectively. The spectral shifts are interpreted to be an indication of a charge, or Stark phenomena, effect on the excited state molecule, as supported by ab initio calculations. To check whether the amino acid remains charged over the temperature range, the infrared spectrum of alanine was monitored over the entire range of temperature. The ratio of infrared absorption characteristic of carboxylate/carbonyl was constant in glycerol/water and sugar glass, which indicates that the charge was retained. Tryptophan buried in proteins, namely calcium parvalbumin from cod and aldolase from rabbit, showed temperature profiles of the fluorescence spectra that were largely independent of the solvent (glycerol/water or sugar glass) and temperature whereas the fluorescence and phosphorescence yields were dependent. The results demonstrate how the rich information found in tryptophan luminescence can provide information on the dipolar nature and dynamics of the matrix.

Animals↗

PubMed2005

Cofilin increases the torsional flexibility and dynamics of actin filaments.

We have measured the effects of cofilin on the conformation and dynamics of actin filaments labeled at Cys374 with erythrosin-iodoacetemide (ErIA), using time-resolved phosphorescence anisotropy (TPA). Cofilin quenches the phosphorescence intensity of actin-bound ErIA, indicating that binding changes the local environment of the probe. The cofilin concentration-dependence of the phosphorescence intensity is sigmoidal, consistent with cooperative actin filament binding. Model-independent analysis of the anisotropies indicates that cofilin increases the rates of the microsecond rotational motions of actin. In contrast to the reduction in phosphorescence intensity, the changes in the rates of rotational motions display non-nearest-neighbor cooperative interactions and saturate at substoichiometric cofilin binding densities. Detailed analysis of the TPA decays indicates that cofilin decreases the torsional rigidity (C) of actin, increasing the thermally driven root-mean-square torsional angle between adjacent filament subunits from approximately 4 degrees (C = 2.30 x 10(-27) Nm2 radian(-1)) to approximately 17 degrees (C = 0.13 x 10(-27) Nm2 radian(-1)) at 25 degrees C. We favor a mechanism in which cofilin binding shifts the equilibrium between thermal ErIA-actin filament conformers, and facilitates two distinct structural changes in actin. One is local in nature, which affects the structure of actin's C terminus and is likely to mediate nearest-neighbor cooperative binding and filament severing. The second is a change in the internal dynamics of actin, which displays non-nearest-neighbor cooperativity and increases the torsional flexibility of filaments. The long-range effects of cofilin on the torsional dynamics of actin may accelerate P(i) release from filaments and modulate interactions with other regulatory actin filament binding proteins.

Actin Cytoskeleton↗

PubMed1993

Excited states of tryptophan in cod parvalbumin. Identification of a short-lived emitting triplet state at room temperature.

The fluorescence and phosphorescence spectra of model indole compounds and of cod parvalbumin III, a protein containing a single tryptophan and no tyrosine, were examined in the time scale ranging from subnanoseconds to milliseconds at 25 degrees C in aqueous buffer. For both Ca- bound and Ca-free parvalbumin and for model indole compounds that contained a proton donor, a phosphorescent species emitting at 450 nm with a lifetime of approximately 20-40 ns could be identified. A longer-lived phosphorescence is also apparent; it has approximately the same absorption and emission spectrum as the short-lived triplet molecule. For Ca parvalbumin, the decay of the long-lived triplet tryptophan is roughly exponential with a lifetime of 4.7 ms at 25 degrees C whereas for N-acetyltryptophanamide in aqueous buffer the decay lifetime was 30 microseconds. In contrast, the lifetime of the long-lived tryptophan species is much shorter in the Ca-free protein compared with Ca parvalbumin, and the decay shows complex nonexponential kinetics over the entire time range from 100 ns to 1 ms. It is concluded that the photochemistry of tryptophan must take into account the existence of two excited triplet species and that there are quenching moieties within the protein matrix that decrease the phosphorescence yield in a dynamic manner for the Ca-depleted parvalbumin. In contrast, for Ca parvalbumin, the tryptophan site is rigid on the time scale of milliseconds.

Animals↗

PubMed1998

Spatial measurement of oxygen levels during photodynamic therapy using time-resolved optical spectroscopy.

Tissue oxygenation is one of the key dosimetric factors involved in the application of photodynamic therapy (PDT). However, quantitative studies of oxygenation levels at and surrounding the treatment site have been lacking both before, during and after treatment. With the recent development of sensitive, non-invasive, optical spectroscopic techniques based on oxygen-dependent phosphorescence quenching of probe compounds, oxygenation levels can now be measured quantitatively at selected sites with spatial resolution on the millimeter scale. We present results using the phosphorescent compound, palladium meso-tetra(carboxyphenyl)porphine, for measurement of in vivo microvascular oxygen tensions in rat liver during PDT. Time-resolved phosphorescence detection was carried out using fibre-optic sensoring, and oxygen tensions were determined from the phosphorescence lifetimes using Stern-Volmer analysis. During PDT treatment using 5-aminolaevulinic (ALA) acid-induced protoporphyrin IX (PPIX) with a 50 mg/kg ALA dose, oxygen levels near the irradiation fibre placed on the surface of the liver showed a significant decrease by a factor of ten from 20 to 2 torr after an energy dose of 60 J using 100 mW at 635 nm. Areas farther from the treatment site which were exposed to lower light doses exhibited lower reductions in oxygen levels. This spectroscopic technique is a highly sensitive means of investigating tissue oxygenation during and after treatment, and should help not only to advance the understanding of hypoxia and microvascular damage in the PDT mechanism but also contribute to improving the dosimetry of PDT.

Aminolevulinic Acid↗

PubMed2002

Measurement of tumor oxygenation using new frequency domain phosphorometers.

Oxygen dependent quenching of phosphorescence allows for non-invasive measurements of oxygen in tissue. We have designed and constructed a novel multi-frequency instrument for measurement of phosphorescence lifetimes and developed algorithms for determining the distribution of oxygen (oxygen histogram) in the microvasculature of tissue with good temporal resolution (Vinogradov et al., 2002, Compar. Biochem. A, these proceedings). This technology, in combination with a new water soluble near infra red phosphor (Oxyphor G2), was used to examine the oxygenation of subcutaneous Q7 tumors grown on the flank of Buffalo rats and their response to giving the rats oxygen or carbogen to breathe. Phosphorescence was measured using excitation at 635 nm and emission at >700 nm (the phosphorescence maximum is near 800 nm). The excitation and collection light guides were placed on the surface of the skin of the anesthetized animals separated by approximately 0.8 cm. A 6 x 6 or 7 x 7 grid (approx. 4 cm x 4 cm) was drawn on the flank and oxygen histograms were measured in each square, providing 'images' of the oxygen distribution in the tissue. This procedure determines the tissue oxygen distribution at each position in the grid. Regions of relative hypoxia (associated with the tumor) can be readily localized and the extent of hypoxia quantitatively evaluated.

Algorithms↗

PubMed1991

Optically detected triplet-state magnetic resonance studies of the DNA complexes of the bisquinoline analogue of echinomycin.

The polymeric DNA and model duplex oligonucleotide complexes of the bisquinoline analogue of echinomycin (2QN) have been studied by optical detection of triplet-state magnetic resonance (ODMR) spectroscopy, with the quinoline chromophores of the drug used as intrinsic probes. Plots of ODMR transition frequencies versus monitored wavelength revealed heterogeneity in the phosphorescence emission of 2QN which was ascribed to the presence of a major and minor conformation of the drug in aqueous solutions (referred to as the red and blue forms of 2QN, respectively, in this report). ODMR results, in conjunction with findings from low-temperature phosphorescence investigations, indicate that the quinoline chromophores of the major (red) form of 2QN are involved in aromatic stacking interactions in complexes with the natural DNAs from Escherichia coli, Micrococcus lysodeikticus, Clostridium perfringens, and calf thymus as evidenced by red shifts in the phosphorescence 0,0-band of the drug, reductions in the phosphorescence lifetime and zero-field splitting (zfs) D and E parameters, and polarity reversals of the ODMR slow passage signals upon complex formation between the analogue and DNA. The polarity reversals, which reflect shifts in the triplet-state sublevel populations induced by complex formation, apparently result from changes in the triplet sublevel decay constants upon binding to the natural DNAs. The 2QN complexes of 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(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 self-complementary oligonucleotides d(ACGT)2, d(TCGA)2, and d(ACGTACGT)2 were also investigated. The extent of reduction of the zfs D parameter (delta D) for the major form of 2QN upon complex formation with the polymeric DNAs was found to scale linearly with the standard free energy of the drug-DNA interaction (delta G degrees) calculated from previously reported binding studies for these targets [Fox, K. R., et al. (1980) Biochem. J. 191, 729-740]. This relationship between spectroscopic and thermodynamic properties of the 2QN-polynucleotide complexes is a consequence of the effects of base stacking interactions on the electronic states of the intercalator, which were postulated to arise from second-order shifts of the ground-state and the triplet-state energies of the complex on the basis of a modification of the solvent effect theory of van Egmond et al. [(1975) Chem. Phys. Lett. 34, 423-426].

Animals↗

PubMed1992

Characterization of tryptophan and coenzyme luminescence in tryptophan synthase from Salmonella typhimurium.

Tryptophan synthase from Salmonella typhimurium is a bifunctional alpha 2 beta 2 complex that catalyzes the formation of L-tryptophan. We have characterized over the temperature range from 160 to 293 K the fluorescence and phosphorescence properties of the single tryptophan present at position 177 of the beta-subunit and of the pyridoxal 5'-phosphate bound through a Schiff's base in the beta-active site. The comparison between the fluorescence of the pyridoxal phosphate bound either to the protein or to valine free in solution indicates substantial protection for the coenzyme against thermal quenching and a greater intensity of the ketoenamine tautomer band. Trp-177 is highly luminescent, and its proximity to the pyridoxal moiety leads to an over 50% quenching of its fluorescence with both reduced and native coenzyme. The Trp phosphorescence spectrum possesses a narrow, well-defined, 0-0 vibrational band centered at 418.5 nm, a wavelength that indicates strong polar interactions with neighboring charges. The observation of delayed fluorescence in the native complex implies that the excited triplet state is involved in a process of triplet-singlet energy transfer to the ketoenamine tautomer. The rate of energy transfer, heterogeneous in low-temperature glasses with rate constants of 2.26 and 0.07 s-1, becomes homogeneous in fluid solutions as the coenzyme tautomer interconversion is likely faster than the phosphorescence decay. In both apo- and holo-alpha 2 beta 2, the phosphorescence from Trp-177 is long-lived even at ambient temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗