Stephen J. Atherton

PHOTOPHYSICAL STUDIES ON HUMAN RETINAL LIPOFUSCIN

Fluorescent material generated in the human retina accumulates within lipofuscin granules of the retinal pigment epithelium (RPE) during aging. Its presence has been suggested to contributed to various diseases including age‐related macular degeneration. Because this material absorbs light at wave lengths as long as 550 nm, photophysical studies were performed to determine whether lipofuscin could contribute to light damage and to determine if its composition is similar to a synthetically prepared lipofuscin. Time‐resolved experiments were performed to monitor (1) fluorescence decay, (2) the UV‐visible absorption of longer‐lived excited states and (3) the formation and decay of singlet oxygen at 1270 nm. Steady‐state and time‐resolved fluorescence studies indicate that human and synthetic lipofuscin have fluorophores in common. Time‐resolved absorption experiments on human retinal lipofuscin and synthetic lipofuscin showed the presence of at least two transient species, one absorbing at 430 nm (lifetime caμs) and a second absorbing at 580 nm, which decays via second order kinetics. In addition, there is a third absorbing species stable to several hundred milliseconds. The transient species at 430 nm is quenched by oxygen, suggesting that it is a triplet state. Subsequent studies showed the formation of singlet oxygen, which was monitored by its phosphorescence decay at 1270 nm. These studies demonstrate that lipofuscin can act as a sensitizer for the generation of reactive oxygen species that may contribute to the age‐related decline of RPE function and blue light damage.

PHOTOCHEMICAL AND PHOTOPHYSICAL STUDIES ON HUMAN LENS CONSTITUENTS

Abstract— –The young human lens contains species (3‐hydroxy kynurenine; 3‐HK and its glucoside; 3‐HKG) which absorb most light between 300 and 400 nm. Photochemical studies have indicated that these compounds are relatively inefficient sensitizers of lens proteins. An investigation of the fluorescent properties of 3‐HKG indicate that it contains a fast deactivation pathway (ps) which would be expected to have minimal photochemical effect on the integrity of the lens. Further photophysical studies on 3‐HK indicates that it has an even faster fluorescent lifetime (< 10 ps) with a much lower quantum yield of fluorescence (0.001 vs 0.03 for 3‐HKG).

TIME RESOLVED SPECTROSCOPIC STUDIES ON THE INTACT HUMAN LENS

Abstract— The human lens is continually under photooxidative stress from ambient radiation. In the young lens the major absorbing (between300–400 nm) species is the glucoside of 3‐hydroxy kynurenine. Using time resolved fluorescence spectroscopy on both the isolated compound and the intact human lens, the first excited singlet state of this compound is shown to have fast (ps) decay processes. This would tend to minimize damage to lens constituents because there would be little time for energy transfer into more harmful channels. Thus, this compound appears to act as a protection for the retina. With aging, human lens proteins become yellow with absorptions out to 450 nm. Time resolved diffuse reflectance spectroscopic studies on intact older human lenses showed that excitation (355 nm) resulted in the formation of long lived (microseconds) transient species with an absorption maximum at ca 490 nm. Similar spectra were obtained from two model systems used to explain age related changes in human lens proteins.

In vivo AND PHOTOPHYSICAL STUDIES ON PHOTOOXIDATIVE DAMAGE TO LENS PROTEINS AND THEIR PROTECTION BY RADIOPROTECTORS

Abstract— Photooxidation, whether initiated by an endogenous or exogenous sensitizer, is an important mechanism in light induced damage to the lens. One of the substrates for this damage is lens protein. A porphyrin sensitizer which binds to lens proteins [mesotetra(p‐sulfonatophenyl) porphyrin (TPPS)] was found to photooxidize Skh‐2 pigmented mice lens protein in vivo. Uroporphyrin, a model for a non‐binding photosensitizer, did not induce photooxidative damage to the mouse lens.

A pulse radiolysis study of the reactions of 3-hydroxykynurenine and kynurenine with oxidizing and reducing radicals

Pulse radiolysis has been used to study the reactions of 3-hydroxykynurenine and kynurenine with solvated electrons, superoxide radicals, hydroxyl radicals and azide radicals. Both 3-hydroxykynurenine and kynurenine react with solvated electrons with diffusion controlled rate constants (k = 2.5 x 10(10) M-1 s-1 and 2.3 x 10(10) M-1s-1, respectively). Neither compound was observed to react with superoxide radicals under our experimental conditions, an upper limit of 1.2 x 10(5) M-1s-1 for the rate constant of this reaction was estimated for both compounds. However, we do observe that a stable product of autooxidation of 3-hydroxy-kynurenine reacts with superoxide radicals and we calculate a lower limit for the rate of this reaction of 5.8 x 10(6) M-1s-1. Reactions of 3-hydroxykynurenine and kynurenine with hydroxyl radicals proceed with diffusion controlled rate constants (1.2 x 10(10) M-1 s-1 and 1.3 x 10(10) M-1 s-1, respectively). The measured values for the rate constants for reaction of 3-hydroxykynurenine and kynurenine with azide radicals are 2.1 x 10(10) M-1s-1 and 4.8 x 10(9) M-1 s-1, respectively. The differences in these rate constants are attributed to differences in the measured oxidation potentials for 3-hydroxykynurenine (+1.0 V vs. NHE) and kynurenine (+1.15 V vs. NHE).

PHOTOPHYSICAL STUDIES ON THE BINDING OF TETRASULFONATOPHENYLPORPHYRIN TO LENS PROTEINS

Abstract— Previous studies have shown that mesotetra(p‐sulfonatophenyl)porphine (TPPS) binds to lens proteins. This characteristic should increase the residence time of the sensitizer in the lens and therefore enhance the probability of inducing photooxidative damage to that tisue in vivo. Subsequent in vivo studies have verified that contention. The present studies were performed to determine the effect of such binding on the spectroscopy and photophysics of the porphyrins.

DETECTION OF PORPHYRIN EXCITED STATES IN THE INTACT BOVINE LENS

Previous steady state and time resolved spectroscopic studies on porphyrins have shown that the triplet lifetimes of those sensitizers that bind to lens proteins are lengthened by several orders of magnitude. Presented here is an extension of this experiment to measure these transients in an intact bovine lens. As demonstrated by steady state fluorescence spectroscopy and flash photolysis, mesotetra (p‐sulfonatophenyl)porphyrin (TPPS) binds to lens proteins. In air‐saturated aqueous solution, TPPS has a triplet lifetime of 2 μs. In an intact bovine lens the triplet state decayed via biexponential kinetics with lifetimes of 0.16 and 1.6 μs. In addition to a lengthening of the lifetime there was a red shift in the triplet transient spectra of10–20 nm of the porphyrin in the intact lenses.

Age-related changes in the human lens as monitored by detection of porphyrin excited states.

Abstract— Previous studies have shown that the triplet state lifetimes of various porphyrins are increased by several orders of magnitude when they are bound to lens proteins. Flash photolysis studies of me‐sotetra (p‐sulfonatophenyl)porphyrin (TPPS) on intact bovine lenses indicated a biexponential decay of the triplet state with lifetimes of 160 μs and 1.6 ms. Here we extend those measurements to TPPS associated with intact human lenses. Steady‐state fluorescence measurements indicate that TPPS binds to both young and old human lenses. In an intact young human lens, the TPPS triplet state is observed to decay biexponentially with lifetimes of 50 and 680 μs. As the age of the lens increases, the lifetime of the shorter‐lived component lengthens while that of the longer‐lived component decreases slightly. In older human lenses, the two lifetimes coalesce and the triplet decay exhibits purely monoexponential behavior. These photophysical characteristics apparently are due to age‐related modification(s) of the protein in the human lens resulting in an increasingly more homogeneous environment around the porphyrin.

THE EFFECT OF MERCURIC IONS ON THE EXCITED STATES OF DNA‐INTERCALATED ETHIDIUM BROMIDE

The first excited triplet state of DNA‐intercalated ethidium bromide is produced with a quantum yield of 0.010.002 on irradiation at 532 nm. A difference extinction coefficient of 1.50.2103 m2 mol−1 is measured for the triplet state at 380 nm. Mercuric ions quench the first excited singlet state of DNA‐intercalated ethidium bromide via induced spin orbit coupling to give an increased yield of ethidium triplet states. The same mercuric ion that quenches the singlet state then quenches the triplet state, via the same mechanism, with a rate constant of ca 3.5103 s−1. An upper limit for the rate of detachment of Hg2+ from its binding site in DNA may be fixed at ca 103 s−1.

LASER FLASH PHOTOLYSIS STUDIES OF DNA-COMPLEXED ETHIDIUM BROMIDE

Abstract— Laser flash photolysis studies of DNA‐complexed ethidium bromide were undertaken. We have observed a singlet‐singlet (S1‐Sn) absorption process for DNA‐complexed ethidium bromide. The observed lowest singlet excited state lifetime was 21 ± 2 ns. The molar difference extinction coefficient was measured to be 2.4 ± 0.4 × 103M‐1 cm‐1 at 370 nm. The assignment of this transition was confirmed by time resolved fluorescence measurements.