Jeffrey R. Kanofsky

QUENCHING OF SINGLET OXYGEN BY HUMAN PLASMA

Abstract— Direct measurements of the decay of singlet oxygen phosphorescence at 1270 nm were made in human plasma diluted with various amounts of deuterium oxide. The Stern‐Volmer plot of the singlet oxygen lifetimes was linear up to 15% plasma concentration (vol/vol). Extrapolation of these measurements to 100% plasma content gave a singlet oxygen lifetime of 1.04 ± 0.03 JJLS in human plasma. Biological molecules accounted for 77% of the total singlet oxygen quenching while water accounted for 23% of the quenching. The contributions of various types of biological molecules to the total singlet oxygen quenching were calculated from their plasma concentrations and their quenching constants. Plasma proteins quenched most of the singlet oxygen. Uric acid also quenched a significant amount of singlet oxygen (12%). Tocopherols, carotenoids, ascorbic acid and bilirubin made only small contributions to the total singlet oxygen quenching (=s 4%).

QUENCHING OF SINGLET OXYGEN BY BIOMOLECULES FROM L1210 LEUKEMIA CELLS

Abstract— Singlet oxygen lifetimes for detergent‐dispersed L1210 leukemia cells in deuterium oxide buffer were measured by following the decay of 1270 nm phosphorescence. Four photosensitizers and two detergents were studied. Stern‐Volmer plots were linear over the cell concentration range studied (0–107 cells/mL). The singlet‐oxygen quenching constants obtained depended somewhat upon the specific combination of detergent and photosensitizer used. Extrapolation of the singlet‐oxygen lifetime data to “100%” cell concentration (1.39 ± 0.04 × 109 cells/mL) and correction for the contribution of the water solvent gave a singlet‐oxygen lifetime between 0.17 and 0.32 us for the L1210 leukemia cell. The theoretical contributions of various types of biological molecules within the L1210 cell to the total singlet‐oxygen quenching were calculated from their concentrations and their quenching constants. These calculations suggest that proteins will quench most of the singlet‐oxygen. Only about 7% of the singlet‐oxygen is quenched by water.

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.

QUENCHING OF SINGLET OXYGEN BY HUMAN RED CELL GHOSTS

Abstract— 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 μs. 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.

Singlet-oxygen Generation from A2E¶

Abstract Singlet-oxygen generation was measured in solutions containing equilibrium mixtures of the retinal lipofuscins, 2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E,7E-octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E-hexatrienyl]-pyridinium (A2E) and double bond isomer of A2E (iso-A2E), using steady-state irradiation and using cholesterol as a singlet-oxygen trap. The amount of singlet oxygen generated by equilibrium mixtures of A2E and iso-A2E was compared with that generated by tetraphenylporphine (TPP) under the same conditions. Studies were carried out in ethanol-d6, acetone-d6, 80% cyclohexane-d12–20% acetone-d6 (vol/vol) and hexafluorobenzene. Using 420 nm irradiation and assuming a singlet-oxygen quantum yield of 0.60 ± 0.12 for TPP, the singlet-oxygen quantum yields were 0.8 ± 0.3 × 10−3, 1.2 ± 0.4 × 10−3, 2 ± 1 × 10−3 and 4 ± 1 × 10−3, respectively. In acetone-d6, the quantum yields were smaller at longer wavelengths, with values of 0.3 ± 0.1 × 10−3 and 0.4 ± 0.2 × 10−3 at 461 and 493 nm, respectively. Singlet-oxygen generation was greatest in solvents with the lowest dielectric constants. In view of the relatively small quantum yields, the contribution of singlet-oxygen generation to the phototoxic properties of A2E and of iso-A2E will require further study.

Direct observation of singlet oxygen phosphorescence at 1270 nm from L1210 leukemia cells exposed to polyporphyrin and light

Near-infrared emission (1170-1475 nm) was studied from L1210 leukemia cells incubated with polyporphyrin (fractionated hematoporphyrin derivative), suspended in deuterium oxide buffer, and then exposed to light. Following pulsed laser excitation, the near-infrared emission decayed in two phases. The first phase of the emission (0-2 microseconds) was principally due to polyporphyrin fluorescence. The second phase of the emission (20-90 microseconds) was due mainly to singlet oxygen. Evidence supporting the assignment of the second phase emission to singlet oxygen included a spectral analysis showing a peak near 1270 nm and reductions in the second phase emission caused by the singlet oxygen quenchers, histidine, carnosine, and water. The second phase emission decayed in a biexponential manner with lifetimes of 4.5 +/- 0.5 and 49 +/- 4 microseconds. Most of the singlet oxygen in the second phase emission was likely due to singlet oxygen that was generated near the surface of the L1210 leukemia cells and then diffused into the deuterium oxide buffer. Direct measurements of singlet oxygen phosphorescence at 1270 nm may prove to be a useful analytical technique for studying photochemical generation of singlet oxygen in cultured cells.

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 (1O2) detection in vivo. The in vivo detection of 1O2 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 1O2 at a subcellular level and more complex mathematical models for interpreting the kinetics of 1O2 emission from living cells. It is now recognized that oxygen consumption, photosensitizer bleaching, oxidation of biological molecules and diffusion of 1O2 can significantly change the kinetics of 1O2 emission from living cells.

SINGLET OXYGEN GENERATION FROM LIPOSOMES: A COMPARISON OF TIME-RESOLVED 1270 NM EMISSION WITH SINGLET-OXYGEN KINETICS CALCULATED FROM A ONE DIMENSIONAL MODEL OF SINGLET-OXYGEN DIFFUSION and QUENCHING

Abstract— Time‐resolved measurements of 1270 nm singlet‐oxygen emission following pulsed‐laser excitation were made from unilamellar dimyristoyl 1‐α‐phosphatidylcholine liposomes labeled with zinc phthalocyanine. The effect of the hydrophobic quenchers, β‐carotene and ethyl β‐apo‐8′trans carotenoate, and the hydrophilic quenchers, histidine and methionine, upon the kinetics of the 1270 nm singlet‐oxygen emission was studied. Hydrophobic quenchers principally lowered the intensity of the 1270 nm emission and caused only modest changes in the lifetime of the 1270 nm emission. The decrease in 1270 nm emission caused by hydrophobic quenchers was related to the size of the liposomes. The larger the radius of the liposome, the greater the decrease in 1270 nm emission caused by a given concentration of hydrophobic quencher. In contrast, hydrophilic quenchers principally decreased the lifetime of the 1270 nm emission. The effect of hydrophilic quenchers was independent of the size of the liposomes.

Structural and Environmental Requirements for Quenching of Singlet Oxygen by Cyanine Dyes

Abstract Singlet-oxygen quenching constants were measured for 19 cyanine dyes in acetonitrile. The most efficient quenchers were 1-butyl-2-[2-[3-[(1-butyl-6-chlorobenz[cd]indol-2(1H)-ylidene)ethylidene]-2-chloro-1-cyclohexen-1-yl]ethenyl]-6-chlorobenz[cd]indolium and 6-chloro-2-[2-[3-(6-chloro-1-ethylbenz[cd]indol-2(1H)-ylidene)ethylidene]-2-phenyl-1-cyclopenten-1-yl]ethenyl]-1-ethylbenz[cd]indolium, having quenching constants with diffusion-controlled values of 2.0 ± 0.1 × 1010 and 1.5 ± 0.1 × 1010 M−1 s−1, respectively. There was a trend toward increased quenching constants for cyanine dyes with the absorption band maxima at longer wavelengths. However, the quenching constants correlated better with the oxidation potentials of the cyanine dyes, suggesting that quenching proceeds by charge transfer rather than energy transfer. The quenching constants for 1,1′,3,3,3′,3′-hexamethylindotricarbocyanine perchlorate and 1,1′-diethyl-4,4′-carbocyanine iodide were measured in several solvents as well as in aqueous solutions of detergent micelles. In different solvents, the quenching constants varied by as much as a factor of 50. The quenching constants were largest in solvents with the highest values on the π* scale of Kamlet, Abboud, Abraham and Taft. This was consistent with quenching occurring by charge transfer. Within cells, cyanine dyes concentrate in membrane-bound organelles. The quenching constants were substantial within detergent micelles. To the extent that micelles are models for biological membranes, cyanine dyes may be effective biological singlet-oxygen quenchers.

Cyanine Dyes as Protectors of K562 Cells from Photosensitized Cell Damage

Abstract Several cyanine dyes were found to protect K562 leukemia cells against toxicity mediated by cis-di(4-sulfonatophenyl)diphenylporphine (TPPS2) and light. Most cyanine dyes derived from dimethylindole were better photoprotectors than cyanine dyes with other structures. This correlated with the fact that cyanine dyes derived from dimethylindole were predominately monomeric at millimolar concentrations within K562 cells, while other cyanine dyes formed aggregates. For cyanine dyes that are derived from dimethylindole and have absorption band wavelengths greater than 700 nm, fluorescence-energy transfer from TPPS2 to the cyanine dye was the most important mechanism for photoprotection. There was no spectroscopic evidence for complex formation between the cyanine dyes and TPPS2. The dimethylindole derivative, 1,1′,3,3,3′,3′-hexamethylindodicarbocyanine, was an excellent photoprotector, but a poor quencher of TPPS2 fluorescence and a relatively poor singlet-oxygen quencher. This cyanine dye may act by quenching excited triplet TPPS2. Singlet-oxygen quenching may contribute to the photoprotection provided by cyanine dyes not derived from dimethylindole. Differences in the subcellular distribution of the various cyanine dyes studied may have contributed to the different apparent mechanisms of photoprotection.