Singlet-Oxygen Generation from Liposomes: A Comparison of 6β-Cholesterol Hydroperoxide Formation with Predictions from a One-Dimensional Model of Singlet-Oxygen Diffusion and Quenching

Abstract— We measured 6β-cholesterol hydroperoxide (6β-CHP), a specific singlet-oxygen (O₂(δg)) product, during irradiation of unilamellar dimyristoyl 1-α-phosphatidylcholine liposomes containing cholesterol and zinc phthalocyanine (ZnPC). The effects of liposome size, the hydrophobic (O₂(¹δ_g)) quencher, β-carotene, and hydrophilic O₂(¹δ_g) quenchers upon the amount of 6β-CHP formed were determined and interpreted in terms of a one dimensional model of ₂(¹δ_g) quenching and diffusion. The model correctly predicted (1) that the amount of 6β-CHP was increased with increasing liposome size, (2) that P-carotene was more effective at reducing 6β-CHP formation in 400 nm diameter liposomes than 100 nm diameter liposomes and (3) that the hydrophilic quencher, water, was also more effective in large liposomes than in small liposomes.
The hydrophobic quencher, β-carotene, was more effective at reducing the formation of 6β-CHP than at reducing the 1270 nm O₂(¹δ_g) emission. This difference was found to be due to the size distribution present in the liposome preparations because the difference between the 6β-CHP data and the 1270 nm emission data was much smaller in liposome preparations with a narrow size distribution. When a significant size distribution was present, the 6β-CHP data were weighted more heavily with large-diameter liposomes, while the 1270 nm emission data were weighted more heavily with small-diameter liposomes.     

TIME-RESOLVED STUDIES OF SINGLET-OXYGEN EMISSION FROM L1210 LEUKEMIA CELLS LABELED WITH 5-(N-HEXADECANOYL)AMINO EOSIN. A COMPARISON WITH A ONE-DIMENSIONAL MODEL OF SINGLET-OXYGEN DIFFUSION AND QUENCHING

Abstract— Time-resolved measurements were made of near-infrared emission from 5-( N -hexadecanoyl)amino-eosinlabeled L1210 leukemia cells following pulsed-laser excitation. The cells were suspended in phosphate-buffered saline made with deuterium oxide solvent. A significant fraction of the emission occuring10–80 μs after the laser pulse was due to singlet oxygen. This singlet-oxygen emission is believed to result from singlet oxygen generated near the cell-membrane surface, where 5-( N -hexadecanoyl)amino eosin is known to concentrate, and then diffusing out into the buffer. The intensity and the kinetics of the experimentally observed singlet-oxygen emission were in excellent agreement with the predictions of a theoretical one-dimensional model of singlet-oxygen diffusion and quenching.
During the10–80 μs time period studied, most of the singlet oxygen was located in the buffer. Thus, the use of water-soluble singlet-oxygen quenchers, such as histidine, provide one means of separating the singlet-oxygen emission quenchers, such as histidine, provide one means of separating the singlet-oxygen emission from other sources of light during this time interval.     

SINGLET-OXYGEN GENERATION AT GAS-LIQUID INTERFACES: A SIGNIFICANT ARTIFACT IN THE MEASUREMENT OF SINGLET-OXYGEN YIELDS FROM OZONE-BIOMOLECULE REACTIONS

Abstract— Several ozone-biomolecule reactions have previously been shown to generate singlet oxygen in high yields. For some of these orone-biomolecule reactions, we now show that the apparent singlet-oxygen yields determined from measurements of 1270 nm chemiluminescence were artifactually elevated by production of gas-phase singlet oxygen. The gas-phase singlet oxygen results from the reaction of gas-phase ozone with biomolecules near the surface of the solution. Through the use of a flow system that excludes air from the reaction chamber, accurate singlet-oxygen yields can be obtained. The revised singlet-oxygen yields (mol ¹O₂ per mol O₃) for the reactions of ozone with cysteine, reduced glutathione, NADH, NADPH, human albumin, methionine, uric acid and oxidized glutathione are 0.23 ± 0.02, 0.26 ± 0.02, 0.48 ± 0.04, 0.41 ± 0.01, 0.53 ± 0.06, 1.11 ± 0.04, 0.73 ± 0.05 and 0.75 ± 0.01, respectively. These revised singlet-oxygen yields are still substantial.     

EVIDENCE FOR THE FORMATION OF A CHLOROPHYLL a/ZEAXANTHIN COMPLEX IN LECITHIN LIPOSOMES FROM FLUORESCENCE DECAY KINETICS

The interaction of Chi a with zeaxanthin (Zea), which is an analogue of lutein, has been studied in soya bean lecithin liposomes using the fluorescence of Chi as monitor. The fluorescence emission spectrum at 4.2 K of Chi a showed characteristic changes in the presence of Zea: the emission maximum shifted from 688 nm to 680 nm, and a peak at 731 nm appeared. The fluorescence decay kinetics of Chi a alone could be described by the sum of two exponential components (T₁,≅0.8 ns, T₂≅2.5 ns). In the presence of Zea a component with a long lifetime, T≅5 ns, appeared with a large relative amplitude (40%). This indicated the formation of a Chl a /Zea complex, in which Chl a /Chl a interaction is negligible, presumably because of strong interaction between Chl a and Zea. The fluorescence anisotropy decay kinetics supported the hypothesis of the formation of a large Chl a containing complex in the presence of Zea. A rotational correlation time, φ≅14 ns at 4°C and φ≅21 ns at 30°C, was found, which is distinctly larger than for samples containing Chl a only. We interpret these results as further evidence for a strong interaction between Chl a and Zea in the hydrophobic environment of the lecithin liposomes. This interaction may also occur in the Chl-proteins of the Chi alb light-harvesting complex of plant photosynthesis.     

Singlet Oxygen-Mediated Inactivation of Acetylcholinesterase: A Comparison of Purified Enzyme in Solution and Enzyme Bound to K562 Leukemia Cells

Abstract— We have compared the singlet oxygen-mediated inactivation of acetylcholinesterase (ACE) in solution with the inactivation of ACE on the surface of K562 leukemia cells. In solution, the actions of the singlet-oxygen quenchers, methionine, azide, disodium [ N,N '-ethylene-bis(5-sulfosalicylideneinuninato)]nickelate(II) (Ni-chelate 1) and disodium [( N,N '-2,3-propionic acid)bis(5-sulfosal-icylideneimminato)]nickelate(II) (Ni-chelate 2) could be explained quantitatively by assuming their only mechanism of action was to quench singlet oxygen. The singlet oxygen quenchers, azide, Ni-chelate 1 and Ni-chelate 2, caused smaller inhibitions in the rate of singlet oxygenmediated inactivation of ACE on K562 cells than ACE in solution. The effects of these quenchers and of deuterium oxide were interpreted using a mathematical model of singlet-oxygen quenching and diffusion to estimate the lifetime of singlet oxygen near the cell surface. The azide quenching data and the deuterium-oxide data gave lifetimes of 0.9 ± 0.2 μs and 0.45 ± 0.15 μs, respectively. The increases in ACE inactivation lifetime caused by the nickel chelates were anomalously large. The unexpectedly large quenching due to the nickel chelates may have been due to a nonuniform distribution of the chelates in the cytoplasm with a large concentration of the chelate near the cell membrane.     

ENERGY TRANSFER AMONG THE CHROMOPHORES OF C-PHYCOCYANIN FROM Anabaena variabilis USING STEADY STATE AND TIME-RESOLVED FLUORESCENCE SPECTROSCOPY

Abstract We have investigated the model of energy transfer between sensitizing (s) and fluorescing (f) chromophores for the αβ monomer and for the separated α and β subunits of C-phycocyanin from Anabaena variabilis using fluorescence emission spectroscopy, fluorescence excitation polarization, and picosecond-resolved fluorescence decay kinetics. The fluorescence emission maximum occurs at 640 nm for all samples. The fluorescence excitation polarization is constant ( P = 0.40) across the absorption hand for the α subunit, but it increases across the absorption band towards longer wavelength for the β subunit and the αβ monomer. The fluorescence decay kinetics exhibit two exponential lifetimes of 1.3-1.5 ns and 340-500 ps for the αβ monomer and for the α and β subunit preparations.
We attribute the change in polarization across the absorption band to energy transfer among the three chromophores in the αβ monomer and among the two chromophores in the separated β subunit. The constant, relatively high polarization in the separated a subunit, having only one chromophore, is consistent with the absence of both energy transfer and chromophore rotation. The concentration of the α subunit did not affect the decay kinetics, suggesting that the short lifetime component does not arise from aggregation of the α subunits. The biexponential decay kinetics of the α subunit cannot be explained using the sensitizing-fluorescing model. The possibility of conformational interactions is under investigation.     

Measurement of singlet-oxygen in vivo: progress and pitfalls

This article is a highlight of the paper by Jarvi et al. in this issue of Photochemistry and Photobiology as well as a brief overview of the state of the field of singlet-oxygen ((1) O(2) ) detection in vivo. The in vivo detection of (1) O(2) using its characteristic 1270 nm phosphorescence is technically challenging. Nevertheless, substantial progress has been made in this area. Major advances have included the commercial development of photomultiplier tubes sensitive to 1270 nm light, techniques for spatially resolving the location of (1) O(2) at a subcellular level and more complex mathematical models for interpreting the kinetics of (1) O(2) emission from living cells. It is now recognized that oxygen consumption, photosensitizer bleaching, oxidation of biological molecules and diffusion of (1) O(2) can significantly change the kinetics of (1) O(2) emission from living cells.     

QUENCHING OF SINGLET OXYGEN BY HUMAN RED CELL GHOSTS

Time resolved measurements of singlet oxygen phosphorescence at 1270 nm were made from unsealed red cell ghosts, labeled with 5-(N-hexadecanoyl)aminoeosin and suspended in deuterium oxide buffer. The singlet oxygen emission lifetime was long, 23 +/- 1 microseconds. The lifetime of the singlet oxygen phosphorescence from intact unsealed ghosts was not a measure of the singlet oxygen lifetime within the red cell ghost membrane, however. The prolonged singlet oxygen emission was due to singlet oxygen escaping from the thin membrane into the buffer, since the emission lifetime was significantly shortened by adding azide ion or water to the deuterium oxide buffer. The lifetime of singlet oxygen within the red cell ghosts membrane was estimated by dispersing the ghosts with detergent and then measuring the singlet oxygen lifetime in deuterium oxide buffers containing various dilutions of the dispersed ghosts. Apparent singlet-oxygen quenching constants were measured using four different photosensitizing dyes and two different detergents. The apparent quenching constant was independent of the dye used, but varied significantly with different detergents. Extrapolation of this data to "100%" ghost concentration gave a singlet oxygen lifetime from 24 and 130 ns. A ghost concentration of "100%" was defined as that concentration of red cell ghost molecules which would be contained within a red cell ghost membrane pellet containing no buffer solutions. Most of the singlet oxygen quenching was due to proteins. Lipids extracted from red cell ghosts accounted for only 2-7% of the total singlet oxygen quenching.     

OCCURRENCE OF THE SINGLET-OXYGEN MECHANISM IN PHOTODYNAMIC OXIDATIONS OF GUANOSINE*

Abstract —Photosensitized oxidations of guanosine in aqueous methanol were investigated with a variety of sensitizers, and several experimental tests for the participation of singlet oxygen were examined. It has been shown that the dye-sensitized photooxidation of guanosine proceeds by both singlet-oxygen and Type I mechanisms, and that the efficiency of the singlet-oxygen mechanism is strongly dependent on photosensitizer type.     

PHOTOOXYGENATION OF MAGNESIUM meso-TETRAPHENYLPORPHYRIN*

Abstract— On unsensitized photooxygenation magnesium meso -tetraphenylporphyrin underwent oxidative ring cleavage yielding a bilitriene derivative as the sole product. Kinetic studies by quenching technique using singlet-oxygen quenchers, ß-carotene and α-tocopherol, and by substrate direct disappearance technique (Foote and Ching) indicated that only singlet-oxygen process is involved in the photooxygenation, and that the rate of total consumption of singlet oxygen ( k _Q+ k _R) is 1.0 ± 0.4 times 10⁸ M ^-1s^-1.     

PHOTOSENSITIZED BLEACHING OF β-CAROTENE WITH LIGHT AT 632·8 nm FROM A CONTINUOUS-WAVE GAS LASER

Abstract— β-Carotene was not affected when irradiated with a monochromatic beam of light (632·8 nm) from a continuous-wave gas laser. However, β -carotene undenvent numerous changes when irradiated in the presence of the photosensitizing dye toluidine blue. Initially there was a considerable decrease in the absorbance of the β-carotene, accompanied by small shifts of the absorption maxima to shorter wavelengths as well as the formation of two new peaks at 400 and 375 nm. Ultimately a complete bleaching of the solution was observed. By the use of column and thin layer chromatography up to 13 different compounds including cis-isomers, epoxides and possibly hydroxy compounds were shown to be formed by the photosensitization of the β-carotene. These reactions were found to be oxygen-dependent.     

FLUORESCENCE AND THE LOCATION OF TRYPTOPHAN RESIDUES IN PROTEIN MOLECULES

Abstract— Fluorescence spectra of a number of native and denaturated proteins have been analysed, using spectral band width (ΔΛ), spectral maximum position (Λ_m), fluorescence quenching by external ionic quenchers, lifetime (b), and quantum yield ( q ) and its changes upon denaturation. The results enabled a model of fluorescence properties of tryptophan residues in the proteins to be substantiated by considering the existence of three discrete spectral classes, one buried in nonpolar regions of the protein (Λ_m 330–332 nm, ΔΛ= 48–49nm, q 0.11, τ= 2.1 ns) and two on the surface. One of the latter is completely exposed to water (Λ_m# 350–353 nm, ΔΛ= 59–61 nm, q # 0.2, τ= 5.4 ns); the other is in limited contact with water which is probably immobilized by bonding at the macromolecular surface (Λ_m# 340–342 nm, ΔΛ= 53–55 nm, q # 0.3, = 4.4 ns). Some quantitative predictions from the model, for (a) the fraction of fluorescence that is quenched by ionic quenchers, (b) the mean values of quantum yield, and (c) the mean values of fluorescence lifetime for various proteins, show good concordance with independent experimentally determined values.     

MECHANISM OF PHOTOINACTIVATION OF PLANT PLASMA MEMBRANE ATPASE

Abstract UV radiation at 290 and 365 nm inactivates two forms of the K⁺-stimulated ATPase associated with the plasma membrane of suspension-cultured cells of Rosa damascena . One form is 15 and 36 times more sensitive than the other to 290 and 365 nm, respectively. For both forms, the inactivation requires oxygen, is inhibited by azide and diazobicyclo(2.2.2.2)octane, but not glycerol, and is enhanced up to 7.5 times in deuterium oxide solvent. Inactivation occurs concomitantly with loss of absorbance at 290 nm. Cs⁺ and NO⁻₃, quenchers of tryptophan fluorescence, inhibit inactivation. The results suggest that inactivation involves singlet-oxygen mediated destruction of tryptophans in the ATPases.     

PORPHYRIN-LIPOSOME INTERACTIONS: INFLUENCE OF THE PHYSICO-CHEMICAL PROPERTIES OF THE PHOSPHOLIPID BILAYER

Abstract— The quenching of the fluorescence emitted by hematoporphyrin incorporated into unilamellar liposomes of dipalmitoyl-phosphatidylcholine and dimyristoyl-phosphatidylcholine, was studied by using methylviologen, 9,10-anthraquinone-2,6-disulfonate and 9,10-anthraquinone-2-sulfonate as quenchers, in order to assess how the distribution of the porphyrin and the interaction mode of the various quenchers with the porphyrin is affected by the physico-chemical properties of the vesicles. The results obtained indicate that, below the critical temperature for the phase transition of the lipids, hematoporphyrin is preferentially distributed in the outer lipid monolayer of liposomes of dipalmitoyl-phosphatidylcholine while most hematoporphyrin molecules are located in the inner monolayer in liposomes of dimyristoyl-phosphatidylcholine. This distribution is only slightly changed when the external mean radius of liposomes increases from 26 to 50 nm. The rise of temperature above the critical value for the liquid-gel phase transition causes a shift of the hematoporphyrin molecules toward the inner phospholipid monolayer. This shift is more pronounced in liposomes of dimyristoyl-phosphatidylcholine. Studies on model systems, i.e. neutral and ionic micelles, indicate that methylviologen and anthra-quinone-type quenchers drastically differ in their interaction mechanism with hematoporphyrin. In particular, methylviologen is the only quencher which can discriminate different hematoporphyrin populations in liposomes of dimyristoyl-phosphatidylcholine and dipalmitoyl-phosphatidylcholine in both the liquid and gel phase. Anthraquinone-type quenchers interact with both hematoporphyrin populations when the lipids are in the gel phase. When the lipids are in a fluid state, the quenching occurs only on the external hematoporphyrin population in liposomes of dipalmitoyl-phosphatidylcho-line while in liposomes of dimyristoyl-phosphatidylcholine no discrimination is observed. The influence of the liposomal structure at different temperatures and of the length of the hydrocarbon chains is discussed.     

ROLE OF THE TRYPTOPHAN FLUORESCENT STATE IN THE ULTRAVIOLET-INDUCED INACTIVATION OF β-TRYPSIN*

Abstract— Stern-Volmer quenching constants for β-trypsin at pH 3 were determined for fluorescence quenching by histidine, acrylamide, and nitrate ion. A modified Stern-Volmer plot (Lehrer, 1971) was employed to show that all of the fluorescent tryptophanyl residues of β-trypsin were equally susceptible to quenching by acrylamide at pH 3 when the enzyme was either in its native conformation or denatured in 6 M guanidine hydrochloride (GuHCl). Fluorescence lifetime measurements indicated that acrylamide quenched β-trypsin fluorescence by a purely collisional mechanism. Solvation of tryptophanyl residues of the protein was maximal at 2.5 M GuHCl, as monitored by fluorescence emission wavelength.
Investigations of the ultraviolet-induced inactivation of β-trypsin at 295 nm were performed in the presence of acrylamide at pH 3. The quantum yields for enzyme inactivation and indole destruction (determined using the PDAB reagent) were unchanged upon depopulation of the fluorescent state by 65 per cent, whether the enzyme was in its native conformation or denatured by 6 M GuHCl. It is concluded that the fluorescent state of tryptophanyl residues of β-trypsin is not involved in enzyme inactivation or tryptophan destruction.     

The quenching of CdSe quantum dots photoluminescence by gold nanoparticles in solution

The photoluminescence (PL) of CdSe quantum dots (QD) in aqueous media has been studied in the presence of gold nanoparticles (NP) with different shapes. The steady state PL intensity of CdSe QD (1.5-2 nm in size) is quenched in the presence of gold NP. Picosecond bleach recovery and nanosecond time-resolved luminescence measurements show a faster bleach recovery and decrease in the lifetime of the emitting states of CdSe QD in the presence of quenchers. Surfactant-capped gold nanorods (NR) with aspect ratio of 3 and surfactant-capped and citrate-capped nanospheres (NS) of 12 nm diameter were used as quenchers in order to study the effect of shape and surface charge on the quenching rates. The Stern-Volmer kinetics model is used to examine the observed quenching behavior as a function of the quencher concentration. It was found that the quenching rate of NR is more than 1000 times stronger than that of NS with the same capping material. We also found that the quenching rate decreases as the length of the NR decreases, although the overlap between the CdSe emission and the NR absorption increases. This suggests that the quenching is a result of electron transfer rather than long-range (Forster-type) energy transfer processes. The quenching was attributed to the transfer of electron with energies below the Fermi level of gold to the trap holes of CdSe QD. The observed large difference between NR and NS quenching efficiencies was attributed to the presence of the [110] facets only in the NR, which have higher surface energy.     

CONFORMATIONAL CHANGES OF BOVINE LENS CRYSTALLINS IN A PHOTODYNAMIC SYSTEM

Abstract— Conformational changes of bovine lens crystallins in a photodynamic system generating singlet oxygen, have been investigated. The formation of intersubunit crosslinks was observed in all three classes (α-, β and γ-) of crystallins by irradiation in the presence of the photosensitizer methylene blue. Near-UV circular dichroism (CD) spectra of the crystallins were significantly altered by irradiation under these conditions, indicating changes in tertiary structure but the far-UV CD remained unchanged suggesting that the secondary structure ((β-sheet conformation) remains unchanged. Significant changes in the absorption and fluorescence spectra were also observed. Measurement of total sulfhydryl content showed a decrease of 27%, 50% and 37% for α-, β- and γ-crystallins respectively, after irradiation. Fluorescence lifetime measurements of N-iodoacetyl-N'-(5-sulfo-l-naphthyl)ethylenediamine-labeled crystallins showed a significant decrease of the lifetime of the major decay components of the label bound to sulfhydryl groups of α- and γ-crystallins, but showed no change in the microenvironment of the sulfhydryl groups of β-crystallin. The results are consistent with the microenvironments of the tryptophan and sulfhydryl groups predicted from sequence studies.     

ON THE TRIPLET-TRIPLET ENERGY TRANSFER FROM CHLOROPHYLL TO CAROTENE IN TRITON X 100 MICELLES

Abstract— The kinetics of the triplet-triplet energy transfer of chlorophyll α (Cha) to β carotene (Car) has been investigated in Triton X100 micelles by 353 nm laser flash photolysis. This transfer consists of an intramicellar process involving pigment species located in the same micelle. A kinetic model using a bimolecular treatment leads to a rate constant of the energy transfer in the micellar phase ( k _tm≅ 6 × 10⁸ M ^-1 s^-1) lower than the previously determined values in homogeneous solvents ( k _t≅ 4.6 ≅ 10⁹ M ^-ls^-l); this result shows the high microviscosity of the micellar core. In addition, the apparent bimolecular rate constant ( k _t≅ 5.0 ≅ 10¹⁰ M ^-l s^-1) appears to be an order of magnitude higher than in homogeneous solvents. The lifetime of the carotene triplet state is the same in the hydrophobic core of Triton X100 micelles (τ a = 7.7 μs) as in organic solvents (hexane or carbon disulfide). The transfer yield is controlled by the distribution of chlorophyll and carotene molecules in the micelles.     

CHANGE IN SULFHYDRYL GROUP MICROENVIRONMENT OF CALF LENS α-CRYSTALLIN BY 300 nm LIGHT

Abstract— The effect of 300 nm irradiation on the sulfhydryl groups of calf lens a-crystallin has been investigated by using specific, covalently bound fluorescent sulfhydryl probes 4–(N-iodoacetoxy)ethyl-N-methylamino-7-n-itrobenz-2-o-xa-1,3-d-iazole (IANBD), N-iodoacetyl-N'-(5-s-ulfo-l-naphthyl) ethylene-diamine (1,5 IAEDANS) and 5-i-odoacetamidofluorescein (IAF). The decrease in tryptophan fluorescence with time of irradiation of a-crystallin, is accompanied by a decrease in the fluorescence of the hydrophobic sulfhydryl label IANBD. In addition, the fluorescence of the surface-sulfhydryl label IAF increased in the irradiated a-crystallin. These results indicate that the sulfhydryl groups are in a more exposed (hydrophilic) environment in the irradiated protein than in the control, possibly because of partial unfolding of the protein. This result is confirmed by fluorescence lifetime measurements with IAEDANS. The decay curve of IAEDANS-α-crystallin has a major lifetime of 15.7 ns and a minor one of 24.6 ns. Upon irradiation, the lifetime of the major component decreases to 10.2 ns and that of the minor component to 21.7 ns. Denatured IAEDANS-α-crystallin has a single lifetime of 10.4 ns. These results show that the photoinduced damage to the tryptophan residues of α-crystallin alters the environment of the sulfhydryl groups and induces a change in the tertiary structure of the protein. Proximity of the cysteine residues to tryptophan in the tertiary structure of the protein may be an important determinant of their susceptibility to photoinduced change.     

A fluorescence spectroscopic study of a coagulating protein extracted from Moringa oleifera seeds

The fluorescence studies of coagulating protein extracted from Moringa oleifera seeds have been studied using steady-state intrinsic fluorescence. The fluorescence spectra are dominated by tryptophan emission and the emission peak maximum (lambda(max)=343+ or -2nm) indicated that the tryptophan residue is not located in the hydrophobic core of the protein. Changes in solution pH affected the protein conformation as indicated by changes in the tryptophan fluorescence above pH 9 whereas the ionic strength had minimal effect. The exposure and environments of the tryptophan residue were determined using collisional quenchers.