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PubMed1990

The anaerobic photolysis of lens alpha-crystallin: evidence for triplet state mediated photodamage.

Anaerobic solutions of lens alpha-crystallin were subjected to near-UV (greater than 295 nm) irradiation, and the photoproducts were analyzed by fluorescence and room-temperature phosphorescence spectroscopy. The principal photoproduct was excited maximally at 340 nm, fluoresced maximally at 430 nm, and phosphoresced with an emission maximum at 510 nm. The phosphorescence intensity decay of this species was well fit by a sum of two exponentials with lifetimes of 9.2 ms (78%) and 61 ms (22%); this report is the first demonstration of a long-lived triplet state associated with a protein photolysis product. As reported previously, 3trp* is also long-lived in deoxygenated alpha-crystallin solution at room-temperature (Berger and Vanderkooi, 1989, Biochemistry 28, 5501-5508), hence both tryptophan and photoproduct triplet states are good candidates to mediate photodamage. Photolysis experiments in the presence of agents known to alter the tryptophan triplet yield provide evidence for the importance of triplet-state-mediated photodamage of lens crystallins in anaerobic solution. In 30 mM acrylamide where 3trp*, but not 1trp*, is efficiently quenched, anaerobic solutions exhibited marked resistance to protein photodamage, whereas the photoprotection in aerobic solution was minimal. In D2O, where photoionization is suppressed but triplet states are longer-lived, photodamage was accelerated in anaerobic solution but reduced in aerobic solutions. Finally, the anaerobic photodestruction rate was increased in 500 mM Cs+ solution where the triplet yield is increased by a heavy atom effect.

Anaerobiosis↗

PubMed1993

Characterization of skeletal muscle actin labeled with the triplet probe erythrosin-5-iodoacetamide.

We have labeled rabbit skeletal muscle actin with the triplet probe erythrosin-5-iodoacetamide and characterized the labeled protein. Labeling decreased the critical concentration and lowered the intrinsic viscosity of F-actin filaments; labeled filaments were motile in an in vitro motility assay but were less effective than unlabeled F-actin in activating myosin S1 ATPase activity. In unpolymerized globular actin (G-actin), both the prompt and delayed luminescence were red-shifted from the spectra of the free dye in solution and the fluorescence anisotropy of the label was high (0.356); filament formation red shifted all excitation and emission spectra and increased the fluorescence anisotropy to 0.370. The erythrosin phosphorescence decay was at least biexponential in G-actin with an average lifetime of 99 microseconds while in F-actin the decay was approximately monoexponential with a lifetime of 278 microseconds. These results suggest that the erythrosin dye was bound at the interface between two actin monomers along the two-start helix. The steady-state phosphorescence anisotropy of F-actin was 0.087 at 20 degrees C and the anisotropy increased to approximately 0.16 in immobilized filaments. The phosphorescence anisotropy was also sensitive to binding the physiological ligands phalloidin, cytochalasin B and tropomyosin. This study lays a firm foundation for the use of this triplet probe to study the large-scale molecular dynamics of F-actin.

Actins↗

PubMed1993

Influence of hydration on the internal dynamics of hen egg white lysozyme in the dry state.

Proteins exist in a predominantly aqueous solvent environment. Hydration of the protein surface significantly affects many aspects of the protein's structure and function; these effects may be related to the molecular dynamics of the protein. We have examined the influence of hydration on the internal dynamics of hen egg white lysozyme using room-temperature phosphorescence from the intrinsic tryptophan residues. Powders of lyophilized lysozyme were hydrated in a phosphorimeter using a flow system that allowed for continuous manipulation of relative humidity over the range 0-92%; this system allowed us to directly compare intensity differences that result from changes in hydration. Lysozyme phosphorescence intensity decreased as a function of hydration over the entire relative humidity range; the decrease was not linear but appeared to occur in distinct phases. The phosphorescence intensity decays were multiexponential over the hydration range studied, and hydration had the largest influence on the long lifetime component. These data suggest that the protein exists in multiple, static conformations in the dry state and that water binding to polar (as opposed to charged) sites on the protein surface induces local and/or global softening of the protein structure.

Animals↗

PubMed1996

Photosensitization by norfloxacin is a function of pH.

Norfloxacin is a fluoroquinolone (FQ) antibiotic that has been reported to cause cutaneous photosensitivity in animals and occasionally in humans. We have studied the fluorescence and singlet oxygen (1O2)-generating properties of norfloxacin. Upon UV excitation the drug fluoresces in water, and the relative intensities of two major fluorescence bands at ca 420 and 450 nm are affected by pH. The overall quantum yield of fluorescence (phi F) is also strongly pH dependent: phi F is low in 0.2 N HCl solution (0.2), increasing steeply to 0.12 at pH 4, then gradually decreasing to 0.01 at pH 10. The changes in phi F are accompanied by changes in fluorescence lifetime from 0.6 ns at pH 1 to 1.8 ns at pH 4. Norfloxacin exhibits phosphorescence in low temperature glasses. The formation of a triplet state at room temperature is also suggested by 1O2 phosphorescence in aerobic D2O. This phosphorescence is "self-quenched" by norfloxacin itself with an efficiency that is pH dependent: kq is 7.9 x 10(6) M-1 s-1 at pD 4, decreases to 1.9 x 10(6) M-1 s-1 at pD 7.5 but then increases about 20-fold in alkaline D2O solutions. This quenching causes the observed 1O2 production by norfloxacin (0.1 mM) to show a maximum at around pH 8-9. However, after correction for self-quenching, the quantum yield of 1O2 production (phi 50)y measured by using perinaphthenone as a standard, yielded the following values: phi s0 is about 0.07 in 0.2 N DCI solution, 0.08 at pH 7.5 and then increases smoothly to approximately 0.2 in 0.1 M NaOD solution. The relatively high, unquenched 1O2 production at physiological pH 7.4 (phi s0 approximately 0.08) suggests that 1O2 reactions may play an important role in the cutaneous phototoxicity of norfloxacin and other FQ antibiotics.

Animals↗

PubMed1991

Optical measurements of the dependence of chemoreception on oxygen pressure in the cat carotid body.

The relationship between oxygen pressure (PO2) in the carotid body and carotid sinus nerve discharge was evaluated in the isolated perfused/superfused cat carotid body using the oxygen-dependent quenching of phosphorescence. Images of phosphorescence intensity arising from Pd-coproporphyrin within the microcirculation of the carotid body provided measurements of intravascular PO2. These measurements were substantiated by determining phosphorescence life-time. The carotid body was perfused in the isolated state via the common carotid artery with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered Tyrode solution, pH 7.4, at a constant pressure of 80 mmHg. Superfusion was maintained with similar media equilibrated with 100% argon. PO2 in the exchange vessels was markedly less than that in the perfusate entering the carotid artery, 23 +/- 3 and 45 +/- 3 Torr for normoxic (111 +/- 15 Torr) and hyperoxic (345 +/- 72 Torr) perfusates, respectively. Chemosensory discharge rose slowly in response to a brief interruption of perfusate flow as PO2 steadily declined from either of these capillary PO2 values to approximately 10 Torr. Between approximately 10 and 3 Torr, chemosensory discharge increased strikingly, concomitant with an enhanced rate of oxygen disappearance, from -36 +/- 4 to -69 +/- 13 (92% change) and -28 +/- 3 to -48 +/- 3 (71% change) Torr/s for normoxic and hyperoxic perfusates, respectively. As PO2 fell below approximately 3 Torr, oxygen disappearance slowed and neural activity decayed. Thus the relationships between microvascular PO2 and chemosensory discharge and between oxygen disappearance and neural discharge suggest that oxygen metabolism in the carotid body determines the expression of oxygen chemoreception.

Animals↗

PubMed1991

Effect of hyperventilation on oxygenation of the brain cortex of newborn piglets.

A new phosphorescence imaging method (Rumsey et al. Science Wash. DC 241: 1649-1651, 1988) has been used to continuously monitor the PO2 in the blood of the cerebral cortex of newborn pigs. A window was prepared in the skull and the brain superfused with artificial cerebrospinal fluid. The phosphorescent probe for PO2, Pd-meso-tetra(4-carboxyphenyl)porphine, was injected directly into the systemic blood. The phosphorescence of the probe was imaged, and the lifetimes were measured using flash illumination and a gated video camera. The PO2 in the blood of the veins and capillary beds of the cortex was calculated from the lifetimes. Systemic blood pressure was continuously monitored while the systemic arterial PCO2, PO2, and blood pH were measured periodically. The PO2 in the blood was quantitated for 60- to 200 microns2 regions within the image (from a total field of approximately 3 mm diam). The PO2 in the microvasculature was not uniform across the viewing field but increased or decreased in each region independently of the other regions. Thus at any point in time the PO2 in a region could be substantially above or below the average value. During hyperventilation, which lowered arterial PCO2 and increased pH of the blood, the average PO2 decreased in proportion to the decrease in arterial PCO2. For example, hyperventilation, which decreased arterial PCO2 from its normal value of 40 Torr to 10 Torr, caused a rapid (within 5 min) decrease in PO2 in the blood of capillaries and veins to approximately one-third of normal.

Animals↗

PubMed1993

Microvascular damage in the cortex of cat brain from middle cerebral artery occlusion and reperfusion.

Oxygen-dependent quenching of phosphorescence provides an extraordinarily powerful method for examining the effects of ischemia/hypoxia on the cortex of the brain. Video camera technology has permitted imaging, through a window in the skull, of the phosphorescence of an oxygen probe, Pd meso-tetra-(4-carboxyphenyl)-porphine, bound to albumin in the blood of anesthetized animals. Images of the phosphorescence taken at different times after the flash of excitation light were used to generate high-resolution two-dimensional maps of the oxygen pressure. These maps show that cortical oxygenation is spatially heterogeneous and that there is dynamic time-dependent modulation of regional oxygen pressures. When the middle cerebral artery was occluded, the region for which it supplied blood became hypoxic, the severity of the hypoxia varying among animals. Release after 60 min of occlusion resulted in a rapid rise of the oxygen pressure to above-normal levels followed by onset of a delayed period of hypoxia. This period is characterized by generally low tissue oxygen pressures with local regions of more severe hypoxia. The delayed period of hypoxia appears to result from damage to the microvasculature, and this microvascular damage is proposed to be an important determinant of the extent of irreversible brain damage.

Animals↗

PubMed1995

Diaphragm microvascular plasma PO2 measured in vivo.

Phosphorescence quenching techniques measure microvascular PO2 without direct surgical manipulation of the tissue. At a given arterial PO2, microvascular PO2 reflects the local O2 uptake-to-O2 delivery ratio, i.e., VO2/QO2. We evaluated the potential of phosphorescence quenching to determine microvascular PO2 in the rat costal diaphragm (PdiaO2). PdiaO2 and arterial blood gases were monitored across transient changes of inspired O2 among 21, 10, and 100% and also during hypotensive states evoked by progressive phlebotomy. After a transit delay, PdiaO2 responded rapidly to alterations of inspired and thus arterial PO2, with half times of the response averaging 5-7 s for switches to a lower inspired O2 (i.e., from 21 to 10%, from 100 to 21%, and from 100 to 10%) and also from 10 to 21%. By comparison, half times of the 10 to 100% and 21 to 100% switches were longer [11 s (P = 0.085) and 21 s (P < 0.05), respectively]. Below a mean arterial blood pressure (BP) of 120-130 mmHg, PdiaO2 decreased as a linear function of BP, with these variables being significantly correlated in each instance (n = 5, r = 0.851-0.937, P < 0.01 for all animals). From these results, it appears feasible to measure PdiaO2 in the spontaneously breathing rat in vivo under steady-state and transient conditions. Also, during progressive hypotension, the fall in PdiaO2 is significantly related to falling BP, likely as a consequence of an increased metabolic demand (increased ventilation and diaphragm VO2) concomitant with decreased blood flow. We conclude that phosphorescence quenching techniques offer a powerful tool for assessing diaphragm physiology and pathophysiology.

Animals↗

PubMed1997

Intravascular oxygen distribution in subcutaneous 9L tumors and radiation sensitivity.

Phosphorescence quenching was evaluated as a technique for measuring PO2 in tumors and for determining the effect of increased PO2 on sensitivity of the tumors to radiation. Suspensions of cultured 9L cells or small pieces of solid tumors from 9L cells were injected subcutaneously on the hindquarter of rats, and tumors were grown to between 0.2 and 1.0 cm in diameter. Oxygen-dependent quenching of the phosphorescence of intravenously injected Pd-meso-tetra-(4-carboxyphenyl) porphine was used to image the in vivo distribution of PO2 in the vasculature of small tumors and surrounding tissue. Maps (512 x 480 pixels) of tissue oxygen distribution showed that the PO2 within 9L tumors was low (2-12 Torr) relative to the surrounding muscle tissue (20-40 Torr). When the rats were given 100% oxygen or carbogen (95% O2-5% CO2) to breathe, the PO2 in the tumors increased significantly. This increase was variable among tumors and was greater with carbogen compared with 100% oxygen. Based on irradiation and regrowth studies, carbogen breathing increased the sensitivity of the tumors to radiation. This is consistent with the measured increase in PO2 in the tumor vasculature. It is concluded that phosphorescence quenching can be used for noninvasive determination of the oxygenation of tumors. This method for oxygen measurements has great potential for clinical application in tumor identification and therapy.

Administration, Inhalation↗

PubMed2003

Fluorescence-based cell viability screening assays using water-soluble oxygen probes.

A simple luminescence-based assay for screening the viability of mammalian cells is described, based on the monitoring of cell respiration by means of a phosphorescent water-soluble oxygen probe that responds to changes in the concentration of dissolved oxygen by changing its emission intensity and lifetime. The probe was added at low concentrations (0.3 microM to 0.5 nM) to each sample containing a culture of cells in the wells of a standard 96-well plate. Analysis of oxygen consumption was initiated by applying a layer of mineral oil on top of each sample followed by monitoring of the phosphorescent signal on a prompt or time-resolved fluorescence plate reader. Rates of oxygen uptake could be determined on the basis of kinetic changes of the phosphorescence (initial slopes) and correlated with cell numbers (10(5) to 10(7) cells/mL for FL5.12 lymphoblastic cell line), cell viability, or drug/effector action using appropriate control samples. The assay is cell noninvasive, more simple, robust, and cost-effective than existing microplate-based cell viability assays; is compatible with existing instrumentation; and allows for high-throughput analysis of cell viability.

Apoptosis↗

PubMed2004

Emergency illumination panel that displays different pictures depending on room brightness.

We fabricated an emergency illumination panel by stacking two translucent pictures, one of which was painted with ink and the other with phosphors. When the panel was illuminated by backlight or room light, only the ink-printed picture was visible to the human eye and only the phosphorescent picture was visible in darkness. A two-way mirror was inserted between the two pictures both to prevent super-imposition of the two images and to enhance phosphorescence. This phosphorescent emergency sign acted as an illuminator independently of electric power sources and provided sufficient brightness for reading newspaper headlines.

Journal Article↗

PubMed2004

Changes in the luminescence between dried and wet bacillus spores.

Fluorescence has been suggested as a method with which to detect and identify bacterial spores. To better understand the nature of the fluorescence signal, we observed the intrinsic steady-state fluorescence and phosphorescence spectra of Bacillus globigii (BG) in both dried and aqueous forms. In vitro, dried, and suspension forms of BG were measured at room temperature in 300-600-nm excitation wavelengths. Also, the phosphorescence of dry BG spores was measured at room temperature at 300-600-nm excitation wavelengths. The wet BG spores exhibited a strong maximum in their fluorescence spectrum, with the peak excitation wavelength near 300 nm and emission wavelength near 400 nm. When the BG was dried, this peak shifted to an approximately 450-nm excitation maximum and an 500-nm emission maximum. The difference between the wet and the dry spore fluorescence spectra cannot be explained by the phosphorescence of the dry spores. Other changes must take place when the spores are wet to account for the large changes observed in the spectrum.

Bacillus↗

PubMed2006

Simultaneous fluorometry and phosphorometry of Langendorff perfused rat heart: ex vivo animal studies.

Fluorescence imaging of intrinsic fluorophores of tissue is a powerful method to assess metabolic changes at the cellular and intracellular levels. At the same time, exogenous phosphorescent probes can be used to accurately measure intravascular tissue oxygenation. Heart failure is the leading cause of death in America. A rat heart can potentially model the human heart to study failures or other abnormalities optically. We report simultaneous fluorescence and phosphorescence measurements performed on a rat heart. We have used two different optical systems to acquire fluorescence signals of flavoprotein and nicotinamide adenine dinucleotide--the two intrinsic fluorophores of mitochondria--and the phosphorescence signal of an intravascular oxygen probe to extract intracellular and intravascular metabolism loads, respectively.

Animals↗

PubMed2002

The relationship between corneal oxygen tension and hypoxic corneal edema.

PURPOSE: We sought to develop a direct, noninvasive measurement of tear oxygen tension behind soft contact lenses, and to use the tear oxygen tension data to determine if subjects with greater comeal oxygen consumption experience more comeal edema under hypoxic conditions. The relationship between tear oxygen tension and comeal thickness was also analyzed to determine if thicker comeas have higher metabolic rates. METHODS: Tear oxygen tension measurement was based on the quenching of a phosphorescent dye by oxygen. By coating a soft contact lens with the dye and placing it in the eye, the rate of decay of phosphorescence could be measured and the tear oxygen tension calculated. Corneal oxygen consumption could then be calculated from the tear oxygen tension. Central corneal swelling was measured after 3 hours of hypoxia induced by contact lens wear under closed-eye conditions. RESULTS: Seven right eyes of seven subjects were tested. Baseline central corneal thickness ranged from 517 to 616 microns. After 3 hours of hypoxia, central corneal swelling ranged from 24.5 to 74.3 microns (3.9% to 13.4%). Steady-state open-eye tear PO2 behind the lens varied between 42.7 and 67.1 torr Pearson correlation suggested possible inverse relationship between open-eye tear oxygen tension and central comeal swelling during hypoxia (r = -0.711, p = 0.0734). CONCLUSIONS: Phosphorescent-based measurements provided a measure of tear oxygen tension in human beings. Low tear film oxygen tensions (high corneal metabolic demand) appeared to predict greater amounts of swelling in subjects' corneas. No relation was found between corneal thickness and tear oxygen tension, suggesting that epithelial and endothelial metabolic rates are more important than stromal thickness in determination of comeal oxygen demand.

Contact Lenses, Hydrophilic↗

PubMed1988

The oxygen dependence of mitochondrial oxidative phosphorylation measured by a new optical method for measuring oxygen concentration.

Oxygen-dependent quenching of phosphorescence has been used to measure the dependence of mitochondrial oxidative phosphorylation on oxygen concentration in suspensions of isolated rat liver mitochondria. An instrument has been designed which simultaneously monitors the phosphorescence lifetime of a fluorophor and the reduction of cytochrome c by dual wavelength spectrophotometry. The phosphorescence lifetime method gives very rapid (less than 100 ms) measure of the oxygen concentration (Vanderkooi, J. M., Maniara, G., Green, T. J., and Wilson, D. F. (1987) J. Biol. Chem. 262, 5476-5482) from concentrations characteristic of air-saturated media to as low as 2 x 10(-8) M. The results may be summarized as follows. For well coupled rat liver mitochondria at pH 7.0 and in the presence of ATP, as the oxygen concentration was lowered, increased cytochrome c reduction was observed to begin at oxygen concentrations greater than 20 microM. For mitochondria in the presence of uncoupler, cytochrome c reduction began at oxygen concentrations less than 1.0 microM. The oxygen dependence of reduction of cytochrome c in well coupled mitochondria treated with ATP was strongly dependent on the pH of the suspending medium. Reduction of cytochrome c began at higher oxygen concentrations as the pH was made more alkaline. The oxygen concentration for half-maximal respiratory rates was much larger for well coupled mitochondria treated with ATP (approximately 0.7 microM) than for mitochondria treated with uncoupler (less than 0.1 microM). It is concluded that the oxygen dependence of mitochondrial oxidative phosphorylation is such that mitochondria could function in their proposed role of tissue oxygen sensors for regulation of such diverse functions as local blood flow and electrical activity in the carotid body.

Adenosine Triphosphate↗

PubMed1986

Direct homogeneous phosphoroimmunoassay for carbamazepine in serum.

In this "phosphoroimmunoassay," a phosphorescent label is used: erythrosin (tetraiodofluorescein). Its long-lived phosphorescence was detected after pulsed excitation in a time-resolved luminescence spectrometer. To achieve convenient open-atmosphere phosphorimetry in liquid solution at room temperature, we used sodium sulfite as an "in situ" chemical deoxygenator, to eliminate oxygen quenching of the excited triplet state. Time-resolved detection allows for complete rejection of short-lived background signals, including those from fluorescent or light-scattering components of biological fluids that can interfere in fluoroimmunoassays. We chose the antiepileptic drug carbamazepine (CBZ) and its active metabolite CBZ-10, 11-epoxide (CBZE) to demonstrate the development and validation of nonseparation assays based on quenching the phosphorescence of erythrosin-labeled drug upon binding to antibody. These two compounds cross reacted equally with the antiserum used; hence, we measured the activity of both in patients' sera. Alternatively, simple pre-treatment of the sample with acid destroys CBZE immunoreactivity and enables specific assay of CBZ.

Carbamazepine↗

PubMedDate not supplied

[Use of triplet state exchange disactivation phenomenon for the study of structure and electron conductivity of proteins].

A method for studying protein structure and estimating its electron conducting properties is proposed. The method is based on the kinetic recording of exchange quenching triplet labels and probes phosphorescence by chromophores or paramagnetic centres. It is shown that different types of exchange interactions (spin exchange, exchange energy transfer) between centres with distance R are described approximately by an equation (I = I0 exp-2R/L) where L changes from 0.7 A (absence of electron coupling--system of type I) to 6.5 A (strong electron coupling--system of type II). I (sec-1) corresponds to exchange energy transfer rate constant or exchange integral in the case of spin exchange. Life-times of excited triplet state eosin-isotiocionate labels connected with the terminal NH2-groups of the following preparations were measured by the method of kinetic phosphorescence decay recording: human oxyhemoglobin, methemoglobin, F- and CN-methemoglobin, metmyoglobin, F- and CN-metmyoglobins. The influence of lysozyme of the nitroxyl spin label bound to His-15 group on the phosphorescence spectrum was investigated. The analysis of our and literature data on the exchange interactions between the centres localized on the protein with known structure (hemoglobin, myoglobin, lysozyme, carboangidrase, bacterial ferredoxin) permit us to conclude that in the examined cases the experimental values correspond to model systems of type I and are different from the dependence in systems of type II by 5--15 order. This allows us to use equation (I) for estimation of the distances between the centres on proteins.

Electron Spin Resonance Spectroscopy↗

PubMed1984

Psoralens and ocular effects in man and animals: in vivo monitoring of human ocular and cutaneous manifestations.

Optical spectroscopy (fluorescence, phosphorescence, and electron spin resonance) has demonstrated the presence of 8-methoxypsoralen (8-MOP) and its potential for photobinding (to DNA and proteins) in human and animal ocular lenses. Similar photobinding in other ocular tissues, particularly the retina, has also been shown to occur in the young eye (lens has not yet developed as an effective UVA filter) and in aphakic and pseudophakic eyes. Skin biopsies were performed on a series of patients before, during, and following psoralen photochemotherapy (PUVA). High-resolution phosphorescence spectroscopy of PUVA patients enabled us to monitor the presence of 8-MOP in the skin and blood samples. These data indicated that significantly higher levels of 8-MOP accumulate within the psoriatic skin compared with uninvolved areas and demonstrated the feasibility of monitoring 8-MOP metabolism by this method. A second series of patients who had been on chronic PUVA therapy for many years were also evaluated by this technique and all patients' eyes were monitored by UV slit-lamp densitography with respect to determining lenticular photodamage in "unprotected eyes" compared with protected and untreated controls. The latter data have delineated at least 3 cases of presumptive PUVA cataracts. Phosphorescence assays on lens matter derived from individuals with a clinical diagnosis of PUVA cataracts demonstrated a photoproduct identical with the previously reported triplet noted in the lenses from experimental PUVA-treated animals.

Animals↗