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PubMed1989

Distance dependence of the tryptophan-disulfide interaction at the triplet level from pulsed phosphorescence studies on a model system.

In the present study the distance dependence of tryptophan-disulfide interaction is examined with a view to both utilizing the interaction as a more quantitative indicator of subtle conformational changes in proteins as well as elucidating the interaction mechanism. To examine perturbations specifically at the indole triplet level 2-(3-indolyl)-ethyl phenyl ketone (IEPK) in which excitation is transferred with high efficiency to the triplet state of the indole moiety was employed. Phosphorescence decays of IEPK excited by a laser pulse in 70/30 (vol/vol) ethanolether at 77 K were measured in the presence of various concentrations of simple disulfides. The nonexponential phosphorescence decays arising from a distribution of fixed chromophoreperturber separations and the steady-state quenching of IEPK were accounted for with an exponential dependence of the quenching rate constant with distance. The small effective Bohr radius (0.8 A) that appears in the exponent emphasizes the localized nature of the interaction. Comparison of the triplet quenching rate constant obtained at quencher contact with IEPK to that estimated in proteins suggests a dependence on the triplet energy of the indole moiety and an endothermic nature for the quenching process. The study predicts that in proteins tryptophan-disulfide interactions are very localized in nature and should give rise to detectable anomalous decays only out to 2 A beyond van der Waals contact between the interacting partners.

Computer Graphics↗

PubMed1991

Time resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging.

An optical microscope capable of measuring time resolved luminescence (phosphorescence and delayed fluorescence) images has been developed. The technique employs two phase-locked mechanical choppers and a slow-scan scientific CCD camera attached to a normal fluorescence microscope. The sample is illuminated by a periodic train of light pulses and the image is recorded within a defined time interval after the end of each excitation period. The time resolution discriminates completely against light scattering, reflection, autofluorescence, and extraneous prompt fluorescence, which ordinarily decrease contrast in normal fluorescence microscopy measurements. Time resolved image microscopy produces a high contrast image and particular structures can be emphasized by displaying a new parameter, the ratio of the phosphorescence to fluorescence. Objects differing in luminescence decay rates are easily resolved. The lifetime of the long lived luminescence can be measured at each pixel of the microscope image by analyzing a series of images that differ by a variable time delay. The distribution of luminescence decay rates is displayed directly as an image. Several examples demonstrate the utility of the instrument and the complementarity it offers to conventional fluorescence microscopy.

3T3 Cells↗

PubMed1993

Glycerol effects on protein flexibility: a tryptophan phosphorescence study.

In exploring the dynamic properties of protein structure, numerous studies have focussed on the dependence of structural fluctuations on solvent viscosity, but the emerging picture is still not well defined. Exploiting the sensitivity of the phosphorescence lifetime of tryptophan to the viscosity of its environment we have used the delayed emission as an intrinsic probe of protein flexibility and investigated the effects of glycerol as a viscogenic cosolvent. The phosphorescence lifetime of alcohol dehydrogenase, alkaline phosphatase, apoazurin and RNase T1, as a function of glycerol concentration was studied at various temperatures. Flexibility data, which refer to rather rigid sites of the globular structures, point out that, for some concentration ranges glycerol, effects on the rate of structural fluctuations of alcohol dehydrogenase and RNase T1 do not obey Kramers' a power law on solvent viscosity and emphasize that cosolvent-induced structural changes can be important, even for inner cores of the macromolecule. When the data is analyzed in terms of Kramers' model, for the temperature range 0-30 degrees C one derives frictional coefficients that are relatively large (0.6-0.7) for RNase T1, where the probe is in a flexible region near the surface of the macromolecule and much smaller, less than 0.2, for the rigid sites of the other proteins. For the latter sites the frictional coefficient rises sharply between 40 and 60 degrees C, and its value correlates weakly with molecular parameters such as the depth of burial or the rigidity of a particular site. For RNase T1, coupling to solvent viscosity increases at subzero temperatures, with the coefficient becoming as large as 1 at -20 degrees C. Temperature effects were interpreted by proposing that solvent damping of internal protein motions is particularly effective for low frequency, large amplitude, structural fluctuations yielding highly flexible conformers of the macromolecule.

Alcohol Dehydrogenase↗

PubMed1994

Phosphorescence lifetime analysis with a quadratic programming algorithm for determining quencher distributions in heterogeneous systems.

A new method for analysis of phosphorescence lifetime distributions in heterogeneous systems has been developed. This method is based on decomposition of the data vector to a linearly independent set of exponentials and uses quadratic programming principles for x2 minimization. Solution of the resulting algorithm requires a finite number of calculations (it is not iterative) and is computationally fast and robust. The algorithm has been tested on various simulated decays and for analysis of phosphorescence measurements of experimental systems with descrete distributions of lifetimes. Critical analysis of the effect of signal-to-noise on the resolving capability of the algorithm is presented. This technique is recommended for resolution of the distributions of quencher concentration in heterogeneous samples, of which oxygen distributions in tissue is an important example. Phosphors of practical importance for biological oxygen measurements: Pd-meso-tetra (4-carboxyphenyl) porphyrin (PdTCPP) and Pd-meso-porphyrin (PdMP) have been used to provide experimental test of the algorithm.

Algorithms↗

PubMed1996

Detection of a pH-dependent conformational change in azurin by time-resolved phosphorescence.

Azurin, a blue copper protein from the bacterial species Pseudomonas aeruginosa, contains a single tryptophan residue. Previous fluorescence measurements indicate that this residue is highly constrained and unusually inaccessible to water. In the apoprotein this residue also possesses a long-lived room-temperature phosphorescence (RTP), the nonexponential decay of which can be resolved into two major components associated with lifetimes of 417 and 592 ms, which likely originate from at least two conformations of the protein. The relative weights of these two decay components change with pH in good correlation with a change in protonation of His-35, which has been studied in Cu(II) azurin. Interestingly, the structural changes characterized in earlier work have little effect on the fluorescence decay and appear to occur away from the tryptophan residue. However, in the present work, the two RTP lifetimes suggest conformations with different structural rigidities in the vicinity of the tryptophan residue. The active conformation that predominates below a pH of 5.6 has the shorter lifetime and is less rigid. Phosphorescence decays of several metal derivatives of azurin were also measured and revealed strong similarities to that of apoazurin, indicating that the structural constraints upon the metal-binding site are imposed predominately by the protein.

Apoproteins↗

PubMed1997

Analysis of phosphorescence in heterogeneous systems using distributions of quencher concentration.

A continuous distribution approach, instead of the traditional mono- and multiexponential analysis, for determining quencher concentration in a heterogeneous system has been developed. A mathematical model of phosphorescence decay inside a volume with homogeneous concentration of phosphor and heterogeneous concentration of quencher was formulated to obtain pulse-response fitting functions for four different distributions of quencher concentration: rectangular, normal (Gaussian), gamma, and multimodal. The analysis was applied to parameter estimates of a heterogeneous distribution of oxygen tension (PO2) within a volume. Simulated phosphorescence decay data were randomly generated for different distributions and heterogeneity of PO2 inside the excitation/emission volume, consisting of 200 domains, and then fit with equations developed for the four models. Analysis using a monoexponential fit yielded a systematic error (underestimate) in mean PO2 that increased with the degree of heterogeneity. The fitting procedures based on the continuous distribution approach returned more accurate values for parameters of the generated PO2 distribution than did the monoexponential fit. The parameters of the fit (M = mean; sigma = standard deviation) were investigated as a function of signal-to-noise ratio (SNR = maximum signal amplitude/peak-to-peak noise). The best-fit parameter values were stable when SNR > or = 20. All four fitting models returned accurate values of M and sigma for different PO2 distributions. The ability of our procedures to resolve two different heterogeneous compartments was also demonstrated using a bimodal fitting model. An approximate scheme was formulated to allow calculation of the first moments of a spatial distribution of quencher without specifying the distribution. In addition, a procedure for the recovery of a histogram, representing the quencher concentration distribution, was developed and successfully tested.

Light↗

PubMed1990

Solid-surface room-temperature phosphorescence detection for high-performance liquid chromatography.

In the present study, we have optimized the use of a two nebulizer system for solid-surface room-temperature phosphorescence (SSRTP) as a selective, permanent record detector for high-performance liquid chromatography (HPLC). The chromatographic parameters and the analytical figures of merit of five well known phosphorescent compounds were compared to those obtained by ultraviolet detection. Calibration curves with satisfactory linear dynamic ranges and limits of detection in the nanogram and subnanogram level showed the feasibility of the SSRTP detector for HPLC. In addition, overlapped compounds were individually identified demonstrating that the selectivity of the proposed detector can be a useful feature in case of incomplete chromatographic separations of complex mixtures.

Benzopyrenes↗

PubMed2002

Tryptophan phosphorescence and pressure effects on protein structure.

After a brief introduction of the potentialities of Trp phosphorescence spectroscopy for probing the conformation and flexibility of protein structure, this presentation summarizes the effects of hydrostatic pressure (up to 3 kbar) on the native fold of monomeric and oligomeric proteins as inferred from the variation of the intrinsic phosphorescence lifetime and the oxygen and acrylamide bimolecular quenching rate constants of buried Trp residues. The pressure/temperature response of the globular fold and modulation of its dynamical structure is analyzed both in terms of a reduction of internal cavities and of hydration of the polypeptide. The implications of these findings for the thermodynamic stability of proteins and for the determination of subunit dissociation equilibria under high pressure conditions are also discussed.

Fluorescent Dyes↗

PubMed2000

Oxygen and acrylamide quenching of protein phosphorescence: correlation with protein dynamics.

Oxygen quenching of protein phosphorescence and activation enthalpies for the structural fluctuations underlying O2 and acrylamide diffusion were determined for RNase T1, glyceraldehyde-3-phosphate dehydrogenase and beta-lactoglobulin, which have the phosphorescing residues located in relatively solvent-exposed and flexible regions of the polypeptide. The results, compared with those obtained for proteins characterised by a very rigid environment, established that kqO2 was directly correlated to the flexibility of the protein matrix surrounding the chromophore. While the migration of acrylamide was characterised by delta H(double dagger), which was strongly dependent on the fluidity of the structure about the Trp residue, the values of the activation enthalpies for the oxygen migration of all the proteins studied were rather similar, approximately 10 kcal mol(-1), in spite of the depth of the chromophore and the rigidity of its environment. The implications of these findings for the migration of small solutes inside proteins have been discussed.

Acrylamide↗

PubMed2003

Structure and dynamics of proteins encapsulated in silica hydrogels by Trp phosphorescence.

This report establishes the conditions for monitoring the intrinsic Trp phosphorescence of proteins encapsulated in silica hydrogels and demonstrates the usefulness of the delayed emission for examining potential perturbations of protein structure-dynamics by the silica matrix. Phosphorescence measurements were conducted both in low temperature (140 K) glasses and at ambient temperature on the proteins apo- and Cd-azurin, alkaline phosphatase and liver alcohol dehydrogenase together with the complexes of liver alcohol dehydrogenase with coenzyme analogs ADPR and H(2)NADH. While spectral shifts and broadening indicate that alterations of the Trp microenvironment are more marked on superficial regions of the macromolecule the decay kinetics of deeply buried chromophores show that the internal flexibility of the polypeptide in two out of three cases is significantly affected by silica entrapment. Both the intrinsic lifetime and the bimolecular acrylamide quenching constant confirm that, relative to the aqueous solution, in hydrogels the globular fold is more rigid with azurin, looser with alcohol dehydrogenase and substantially unaltered with alkaline phosphatase. It was also noted that large amplitude structural fluctuations, as those involved in coenzyme binding to alcohol dehydrogenase or thermally activated in alkaline phosphatase, were not restricted by gelation. Common features of the three silica entrapped proteins are pronounced conformational heterogeneity and immobilization of rotational motions of the macromolecule in the long time scale of seconds.

Acrylamides↗

PubMed2002

Recovery of radial PO(2) profiles from phosphorescence quenching measurements in microvessels.

Work by previous investigators has indicated that a substantial amount of oxygen diffuses from the precapillary circulation. These losses imply that there should be radial gradients of oxygen tension (PO(2)) in arterioles, leading to a non-uniform distribution of oxygen within these microvessels. We have employed the phosphorescence quenching method to measure oxygen, allowing us to evaluate the heterogeneity of PO(2) inside short segments of microvessels. The phosphorescence decay curve contains information about the distribution of oxygen within the excited volume and the distribution can be represented as a histogram, by decomposing the decay curve into several components with weights proportional to the volume fraction of plasma with different PO(2), under the condition of a high signal-to-noise ratio. Furthermore, the histogram can be converted into a radial profile of PO(2), based on the assumptions of a circular vascular lumen, axisymmetric distribution of oxygen and monotonic PO(2) profile. Albumin-bound Pd-porphyrin phosphor was infused into the circulation of hamsters and excited by flash illumination at 10 Hz, with a square region of excitation light just covering the entire lumen, (i.e. width of region equaled luminal diameter) of microvessels in the hamster mesentery. A set of 50 curves (5 s of data) was averaged to obtain a decay curve with low noise. Curves were analyzed with the above histogram procedure, and this analysis allowed us to distinguish between PO(2) values originating from intra and extravascular subvolumes. The intravascular PO(2) in these microvessels was very heterogeneous, which could be explained by the existence of significant radial PO(2) gradients. The radial PO(2) gradients were estimated to be approximately 1 mmHg/microm.

Animals↗

PubMed2001

Study of properties on non-protected room temperature phosphorescence and delayed excimer fluorescence of pyrene solution.

A strong and stable room temperature phosphorescence (RTP) and delayed excimer fluorescence signal located at 596 and 475 nm, respectively, can be induced for pyrene solution in the absence of any protective medium only use KI or TlNO3 as a heavy atom perturber (HAP) and Na2SO3 as a deoxygenator. Both lifetimes of RTP and the delayed fluorescence are in the order of X-ms and the intensities are changed with kind and amount of HAP, but the peak positions are same and there is a iso-luminescent point in the emission spectra corresponding to emission at 475 nm and at 596 nm. The optimum conditions and the effects of kind and amount of HAP and organic solvents on luminescence properties of pyrene solution were studies in detail, and the photophysical process in the presence of KI or TlNO3 for phosphorescence and delayed excimer fluorescence emission of pyrene solution was discussed.

Fluorescent Dyes↗

PubMed2002

Room temperature phosphorescence of 6-bromo-2-naphthol in micelles of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) block copolymers and in their mixed aggregates with sodium dodecyl sulfate.

Room temperature phosphorescence (RTP) of 6-bromo-2-naphthol has been investigated in aqueous micellar solutions of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) block copolymers as well as in their mixed aggregates with sodium dodecyl sulfate. RTP of the phosphorophor was enhanced to some extent in the micelles of the block copolymers. However, marked enhancement of RTP was observed in the mixed aggregates. The enhancement of RTP is attributed to effective incorporation of the phosphorophor into the micelles and the aggregates, resulting in suppression of nonradiative deactivation of the phosphorescent state.

Micelles↗

PubMed2002

Studies on properties and application of non-protected room temperature phosphorescence of propranolol.

A direct and simple non-protected room temperature phosphorimetry (NP-RTP) for determine propranolol, which using I- as a heavy atom perturber and sodium sulfite as a deoxygenator, has been developed. The phosphorescence peak wavelength maxima lambda(ex)/lambda(em) = 288/494, 522 nm. The analytical curve of propranolol gives a linear dynamic range of 8.0 x 10(-8)-2.0 x 10(-5) mol l(-1) and a detection limit of 3 x 10(-8) mol l(-1). The influence of I- concentration on RTP lifetime of propranolol was studied and the luminescence kinetic parameters were calculated. It is found that the relation between I- concentration (x) and RTP lifetime (tau) can be expressed as tau = 1.25e(-0.477x) and the rate constants of phosphorescence emission k(p) was 0.800 per ms. The method was applied directly to determination of propranolol in urine and drug tablets with a satisfactory result. The recoveries were 96.6-97.4% and the relative standard deviation was 2% for the 1.00 x 10(-6)-4.00 x 10(-6) mol l(-1) propranolol in spiked urine sample.

Humans↗

PubMed2003

The lowest excited triplet (T1) energies of p-methoxybenzaldehyde and p-cyanobenzaldehyde vapors estimated from the temperature dependence of the T2(n, pi) phosphorescence and the S1(n, pi) fluorescence spectra.

Invisible energy levels of the T1(pi, pi*) state of p-methoxybenzaldehyde (anisaldehyde) and p-cyanobenzaldehyde vapors have been estimated through the temperature dependence of the T2(n, pi*) --> S0 phosphorescence and the S1(n, pi*) --> S0 delayed fluorescence spectra. It is shown that the T1(pi, pi*) levels are located at 900 +/- 100 and 300 +/- 100 cm(-1) below the T2(n, pi*) levels, respectively, for p-methoxybenzaldehyde and p-cyanobenzaldehyde vapors. The estimated T1 energy levels are in good agreement with the phosphorescence origins in rigid glass at 77 K.

Benzaldehydes↗

PubMed2004

Study on aggregation of palladium-porphyrins using room temperature phosphorescence.

This paper reported room temperature phosphorescence (RTP) behaviors of meso-tetra-(4-sulfonatophenyl) porphyrin palladium (Pd-TSPP) and meso-tetra-(4-trimethylaminophenyl) porphyrin palladium (Pd-TAPP) in bovine serum albumin (BSA) medium. It was found that Pd-TSPP self-aggregated with its increasing concentration and hetero-aggregated with Pd-TAPP when they were mixed together. The self-aggregation of Pd-TSPP resulted in the remarkably splitting of excitation spectra because of the strongly excitonic coupling and phosphorescence quenching excited by Soret band, while Q band always kept the increase in intensity. The hetero-aggregation was out of the ground-state interaction stronger than the former one owing to its electrostatic-interaction nature. It was also indicated that inorganic salts like KCl would be an aid to hetero-aggregation. The equilibrium constants of both kinds of aggregation were estimated, namely, K(hom)=1.9 x 10(5) l/mol (homo-aggregation), and K(het)=1.06 x 10(7) l/mol (hetero-aggregation).

Animals↗

PubMed1994

Silica-based room temperature phosphorescence substrates: wide dynamic ranges, low background, and programmable figures of merit.

The use of silica-based substrates for room temperature phosphorescence (RTP) was investigated. Contrary to reports in the literature, bare silica was shown to provide higher sensitivity (S/B) for RTP than filter paper when the specific surface areas of the two substrates were similar. The surface heterogeneity of bare silica resulted in nonlinear, discontinuous response curves for a model analyte, phenanthrene. However, analytically useful calibration curves were obtained on silica thin-layer phases in the presence of coadsorbed surfactant. The dynamic range on the silica substrate was 4 times greater and the background phosphorescence was 2 orders of magnitude lower than filter paper. The programming of figures of merit for silica-based substrates via the silica specific surface area is demonstrated.

Fluorometry↗

PubMed2002

Performance evaluation of the phosphorescent porphyrin label: solid-phase immunoassay of alpha-fetoprotein.

Phosphorescent conjugates of antibodies, neutravidin, and biotin (pentylamine derivative) were synthesized using previously described monofunctional labeling reagent of platinum(II) coproporphyrin-I with isothiocyanate reactive group (PtCP-NCS). These conjugates, which can be considered as standard reagents for a range of bioanalytical applications, were evaluated in solid-phase immunoassay schemes with the clinical analyte a-fetoprotein (AFP). A custom-designed time-resolved phosphorescence plate reader based on a compact and low-cost 532-nm laser and optimized for measurement of porphyrin labels was used. Using optimized tracers, instrumentation and assay protocols, subpicomolar detection limits were obtained both for PtCP label in solution and for AFP in solid-phase immunoassay. This sensitivity is comparable with standard time-resolved fluorescence immunoassays with lanthanide labels. The performance of metalloporphyrin labels, instrumentation, and solid-phase immunoassays as an alternative to the established detection platforms is discussed.

Biotin↗