Fluorescence studies of lens epithelial cells and their constituents.

Steady-state and time-resolved fluorescence measurements have been made of human and rabbit lens epithelial cells and their total soluble protein. Excitation at 350 nm results in broad fluorescence spectra peaking at 450 nm and stretching into the visible past 650 nm. The fluorescence excitation spectra peak around 350 nm. We assign the species responsible for this absorption and fluorescence as NADPH. Because the absorption of near-UV light (300-400 nm) is responsible for cell damage and death, we postulate that excited states of NADPH are implicated in the mechanisms of cell damage. Preirradiation with 355 nm light leads to a loss of NADPH fluorescence but with no change in decay kinetics. Possible mechanisms for cell damage are explored.

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

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 protein. Flash photolysis studies of mesotetra (p-sulfonatophenyl)porphyrin (TPPS) on intact bovine lenses indicated a biexponential decay of the triplet state with lifetimes of 160 microsecond 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 microsecond. 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 order 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.

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 ca 7 microseconds) 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.

Photodissociation and recombination of carbonmonoxy cytochrome oxidase: dynamics from picoseconds to kiloseconds.

The kinetics of the flash-induced photodissociation and rebinding of carbon monoxide in cytochrome aa3-CO have been studied by time-resolved infrared (TRIR) and transient ultraviolet-visible (UV-vis) spectroscopy at room temperature and by Fourier transform infrared (FTIR) spectroscopy at low temperature. The binding of photodissociated CO to CuB+ at room temperature is conclusively established by the TRIR absorption at 2061 cm-1 due to the C-O stretching mode of the CuB(+)-CO complex. These measurements yield a first-order rate constant of (4.7 +/- 0.6) x 10(5) s-1 (t1/2 = 1.5 +/- 0.2 microseconds) for the dissociation of CO from the CuB(+)-CO complex into solution. The rate of rebinding of flash-photodissociated CO to cytochrome a(3)2+ exhibits saturation kinetics at [CO] > 1 mM due to a preequilibrium between CO in solution and the CuB(+)-CO complex (K1 = 87 M-1), followed by transfer of CO to cytochrome a(3)2+ (k2 = 1030 s-1). The CO transfer from CuB to Fe alpha 3 was followed by CO-FTIR between 158 and 179 K and by UV-vis at room temperature. The activation parameters over the temperature range 140-300 K are delta H++ = 10.0 kcal mol-1 and delta S++ = -12.0 cal mol-1 K-1. The value of delta H++ is temperature independent over this range; i.e., delta Cp++ = 0 for CO transfer. Rapid events following photodissociation and preceding rebinding of CO to cytochrome a(3)2+ were observed. An increase in the alpha-band of cytochrome a3 near 615 nm (t1/2 ca. 6 ps) follows the initial femtosecond time-scale events accompanying photodissociation. Subsequently, a decrease is observed in the alpha-band absorbance (t1/2 approximately 1 microsecond) to a value typical of unliganded cytochrome a3. This latter absorbance change appears to occur simultaneously with the loss of CO by CuB+. We ascribe these observations to structural changes at the cytochrome a3 induced by the formation and dissociation of the CuB(+)-CO complex. We suggest that the picosecond binding of photodissociated CO to CuB triggers the release of a ligand L from CuB. We infer that L then binds to cytochrome a3 on the distal side and that this process is directly responsible for the observed alpha-band absorbance changes. We have previously suggested that the transfer of L produces a transient five-coordinate high-spin cytochrome a3 species where the proximal histidine has been replaced by L. When CO binds to the enzyme from solution, these processes are reversed.(ABSTRACT TRUNCATED AT 400 WORDS)

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).

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 0.01 +/- 0.002 on irradiation at 532 nm. A difference extinction coefficient of 1.5 +/- 0.2 x 10(3) 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 3.5 x 10(3) s-1. An upper limit for the rate of detachment of Hg2+ from its binding site in DNA may be fixed at ca 10(3) s-1.

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 microseconds. In an intact bovine lens the triplet state decayed via biexponential kinetics with lifetimes of 0.16 and 1.6 microseconds. In addition to a lengthening of the lifetime there was a red shift in the triplet transient spectra of 10-20 nm of the porphyrin in the intact lenses.

Nature and functional implications of the cytochrome a3 transients after photodissociation of CO-cytochrome oxidase.

Time-resolved electronic absorption, infrared, resonance Raman, and magnetic circular dichroism spectroscopies are applied to characterization of the intermediate that is formed within 20 ps after photodissociation of CO from cytochrome a3 in reduced cytochrome oxidase. This intermediate decays with the same half-life (approximately 1 microseconds) as the post-photodissociation CU+B-CO species previously observed by time-resolved infrared. The transient UV/visible spectra, kinetics, infrared, and Raman evidence suggest that an endogenous ligand is transferred from CuB to Fea3 when CO binds to CuB, forming a cytochrome a3 species with axial ligation that differs from the reduced unliganded enzyme. The time-resolved magnetic circular dichroism results suggest that this transient is high-spin and, therefore, five-coordinate. Thus we infer that the ligand from CuB binds on the distal side of cytochrome a3 and displaces the proximal histidine imidazole. This remarkable mechanistic feature is an additional aspect of the previously proposed "ligand-shuttle" activity of the CuB/Fea3 pair. We speculate as to the identity of the ligand that is transferred between CuB and Fea3 and suggest that the ligand shuttle may play a functional role in redox-linked proton translocation by the enzyme.

In vivo and photophysical studies on photooxidative damage to lens proteins and their protection by radioprotectors.

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. The radioprotector 3-amino-2-hydroxypropyl phosphorothioate (WR-77913) was investigated as an agent to retard or negate in vivo photooxidative damage to the lens. Intraperitoneal injections of WR-77913 prior to irradiation reduced the TPPS induced photodestruction of lens protein in Skh-2 pigmented mice. The mechanism of protection was also investigated. Thiols were found to quench both the triplet state of porphyrins and the reactive intermediate singlet oxygen on the order of 10(5) and 10(6) M-1 s-1 respectively. These are probably not fast enough to explain most of the protection afforded by thiols. An additional mechanism may be the accelerated photobleaching of porphyrins by thiols which protects tissue by reducing the absorptions due to the porphyrins.

Photophysical studies on the binding of tetrasulfonatophenylporphyrin to lens proteins.

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 tissue 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. It was found that the binding of TPPS (1) quenches the fluorescence of lens proteins, (2) causes a shift in the ground state absorption spectra, fluorescence excitation spectra and the triplet excited state spectrum of TPPS to longer wavelengths and (3) results in an increase in the triplet state lifetime of TPPS. In the presence of the isolated crystallins the average triplet lifetime increases in the following order: gamma less than beta less than alpha.

Photochemical and photophysical studies on human lens constituents.

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 phot physical studies on 3-HK indicates that it has an even faster fluorescent lifetime (less than 10 ps) with a much lower quantum yield of fluorescence (0.001 vs 0.03 for 3-HKG). With aging, the human lens proteins undergo numerous changes including a generalized yellowing. These chromophores exhibit a higher quantum yield of fluorescence, an increase in the fluorescent lifetime by 2 orders of magnitude and the formation of two long lived transient species (microsecond). These species might be expected to drastically increase the susceptibility of the human lens to ambient radiation. Based upon quantitative experimental comparisons with 3-HK this does not seem to be the case. Further time resolved studies on old lens proteins indicate that the two transient species are interconnected in that the first transient species is the precursor to the second. The implications of this mechanism on the integrity of the lens and origin of those chromophores is discussed.

Time resolved spectroscopic studies on the intact human lens.

The human lens is continually under photooxidative stress from ambient radiation. In the young lens the major absorbing (between 300-400 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 absorption 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.