A method for detecting 8-methoxypsoralen in the ocular lens.

The use of 8-methoxypsoralen for treating psoriasis could prove hazardous if this photosensitizing agent enters the ocular lens. Phosphorescence spectra of intact rat lenses reveal concentrations of 8-methoxypsoralen on the order 10(-5)M after intraperitoneal injection of 8-methoxypsoralen. There is evidence that this drug can function as a photosensitizing agent, enhancing ultraviolet-induced changes within the lens.

Photodamage to calf lenses in vitro by excimer laser radiation at 308, 337, and 350 nm.

Calf lenses in vitro were irradiated using an excimer laser at wavelengths of 308, 337, and 350 nm for times ranging from 10 minutes to 5 hours. The laser power was 2.0 watts (W) at 308 nm, 0.2 W at 337 nm, and 2.0 W at 350 nm. During irradiations, the visible light transmission (632.8 nm) of the lenses was measured and found to be decreased markedly with 308- or 337-nm irradiation. No change in visible light transmission was observed with irradiation at 350 nm. Irradiated lenses were also compared with dark control lenses by photographic record. Lenses exposed to 308-nm ultraviolet (UV) radiation for 10-30 minutes showed significant yellow-brown pigmentation and colorless opacification compared with dark controls. Lenses exposed to 337-nm UV light showed primarily colorless opacity with little pigment production. Lenses exposed to 350-nm radiation for up to 1 hour were visibly indistinguishable from dark controls. After photolysis, the lenses were separated into water-soluble and insoluble fractions and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Compared with dark controls, UV-exposed lenses (308 or 337 nm) showed loss of 20-30-kilodalton (kD) material and production of higher molecular weight material at 40-60 kD and greater than 100 kD. There was no evidence of such changes after 350-nm exposure. The data gave the following order for the degree of photodamage: 308 nm approximately 5 x 337 nm greater than 20 x 350 nm. An action spectrum for lens damage is presented.

Photosensitization of the lens by 8-methoxypsoralen.

During the past decade, ambient ultraviolet radiation has been implicated in the age-related increase in fluorescence and pigmentation of the human lens nucleus. 8-Methoxypsoralen (8-MOP) (currently in vogue for the treatment of psoriasis) is a well-known photosensitizing agent. This drug lar fluorescence (360/440 nm.) and a change in receiving a single intraperitoneal dose (4 to 8 mg./kg.) of 8-MOP. When such rats are subjected to ambient light or ultraviolet (UV) radiation in vivo, there is an enhancement of lenticular fluorescence (360/440 nm.) and a change in their phosphorescence spectra. In vitro studies on lenses derived from rats given 8-MOP and exposed to monochromatic UV radiation show effects similar to those of the in vivo experiments. The foregoing studies demonstrate that 8-methoxypsoralen enters the lens and can be affected by ambient light as well as UV radiation, resulting in a photosensitized enhancement of lenticular fluorescence and a binding of this photosensitizing agent to macromolecules within the lens.

Picosecond fluorescence decay of lens protein gamma-II crystallin.

The fluorescence decay of tryptophan residues in the bovine lens protein gamma-II crystallin has been measured in aqueous buffer solutions. Results were obtained as a function of emission wavelength, temperature, dissolved oxygen, and denaturing solvent. The protein displayed complex fluorescence decay which fit a biexponential model with a long component (ns) and a short component (few hundred ps). Measured fluorescence quantum yields data for gamma-II crystallin allowed calculation of radiative and non-radiative rate constants. The radiative rate constant was consistent with that observed in other indole derivatives, while the non-radiative rate constant was quite large and accounted for the short lifetime in gamma-II. The temperature dependence of the non-radiative decay in gamma-II crystallin yielded a small activation energy of only 1-2 kcal/mol, compared to 4 kcal/mol for the reference compound NATA whose barrier is known to derive from the rotamer model.

Energetics and possible mechanisms of ion-beam protein tritiation based on AB initio potential surfaces.

Ab initio self-consistent field potential energy surfaces for the approach of T, T2, T+, T3+ and HeT+ to glycine in the gas phase have been determined and this data used to obtain insight into mechanisms of experimental ion-beam protein tritiation processes. Results of these calculations show that the ionic species T+, T3+ and HeT+ can form stable adducts with glycine (Gly) and that each functions as a tritiation agent forming the complex GlyT+. Neutral T and T2 experience a purely repulsive interaction with Gly and do not form an intermediate complex. These neutral species are expected to be less effective tritiation agents than the respective ions, in agreement with experimental observations. The fate of the stable GlyT+ complex is discussed and it is proposed that this species is neutralized by electron capture to give GlyT which spontaneously dissociates to either Gly+T or tritiated glycine (Gly*)+H, with the latter reaction product channel favored statistically. The most likely site of exchange is predicted to be at the amine nitrogen although significance exchange is expected to occur at the alpha-carbon site by a somewhat more complex reaction mechanism.

Concentration dependence of transmission losses in UV-laser irradiated bovine alpha-, beta H-, beta L- and gamma-crystallin solutions.

Experiments with calf lens protein fractions in aqueous buffer solutions at room temperature showed that beta H-, beta L- and gamma-crystallin fractions became opaque following ultraviolet exposure at 308 nm, while the alpha-crystallin fraction remained transparent. Transmission loss, due to UV-irradiation, for all of the crystallin samples was studied in the concentration range of 0.1 mg/mL to 1.0 mg/mL, and for alpha- and gamma-crystallin, in the range up to 5 mg/mL. With increased concentrations of beta H-, beta L- and gamma-crystallin, the rate of opacification increased. However, with alpha-crystallin, the loss of transmission was negligible for all of the concentrations and irradiation times studied. Opacification of the crystallins was accompanied by formation of higher molecular weight insoluble proteins as detected by SDS-PAGE.

The molecular chaperone function of alpha-crystallin is impaired by UV photolysis.

Buffer solutions of the lens protein gamma-crystallin and the enzymes aldolase and liver alcohol dehydrogenase became turbid and formed solid precipitate upon exposure to an elevated temperature of 63 degrees C or to UV radiation at 308 nm. When alpha-crystallin was added to the protein solutions in stoichiometric amounts, heat or UV irradiation did not cause turbidity, or turbidity developed much less rapidly than in the absence of alpha-crystallin. Hence, normal alpha-crystallin functioned as a "molecular chaperone," providing protection against both UV and heat-induced protein aggregation. When alpha-crystallin was preirradiated with UV at 308 nm, its ability to function as a chaperone vis-a-vis both UV and heat-induced aggregation was significantly impaired, but only at relatively high UV doses. A major effect of preirradiation of alpha-crystallin was to cause interpeptide crosslinking among the alpha A2 and alpha B2 subunits of the alpha-crystallin macromolecule. In our experiments alpha-crystallin was exposed to UV doses, which resulted in 0.50 and 90% crosslinking as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. alpha-Crystallin samples that were 50% and 90% crosslinked gave chaperone protection, which was increasingly impaired relative to unirradiated alpha-crystallin. The results are consistent with the notion that UV irradiation of alpha-crystallin results in loss of chaperone binding sites.

The rates of photolysis of the four individual tryptophan residues in UV exposed calf gamma-II crystallin.

Aqueous buffer solutions of the lens protein bovine gamma-II crystallin were irradiated at 295 nm in the presence of dithiothreitol to determine the individual photolysis susceptibilities of the four tryptophan residues. Reverse-phase high performance liquid chromatography was utilized to compare the tryptic peptide maps before and after irradiation. Sequence analysis of collected tryptic peptides showed that the four tryptophans in calf gamma-II crystallin. TRP-42, TRP-68, TRP-131, and TRP-157 appeared in four distinct tryptic peptides. Fluorescence and absorption (diode array) monitoring of the eluting peptides allowed assessment of the changes in peptide absorbance and fluorescence following irradiation. Tryptophan fluorescence losses of (40 +/- 15)%, (17 +/- 4)%, (35 +/- 5)% and (15 +/- 4)% were observed for the peptides containing TRP-42, TRP-68, TRP-131 and TRP-157, respectively. Thus the four tryptophans in calf gamma-II crystallin did not all photolyze at the same rate. The rate differences are presumably related to the microenvironments of the individual tryptophan residues, and this is discussed in terms of the known crystal structure of calf gamma-II crystallin.

Analysis of photo-oxidized amino acids in tryptic peptides of calf lens gamma-II crystallin.

Insoluble and crosslinked proteins and increased pigmentation in the eye lens are features of aging and cataracts. Determining the amino acids which are involved in insolubilization, crosslinking and visible light scattering will shed light on the mechanisms by which cataracts form. Calf lens gamma-II crystallin was irradiated at 295 nm, digested and separated into tryptic peptides. Additional tryptic peptides were found in the digest of irradiated gamma-II which were not present in the dark control digest. These peptides were identified by amino acid sequencing and shown to correspond to expected tryptic fragments of the protein, indicating more facile digestion in the UV-irradiated protein than in dark controls. Amino acid analysis of the irradiated protein and peptides showed losses of histidine, methionine and cysteine residues as compared to control samples. Tryptophan, which is not detected by amino acid analysis, was also found to be reactive since losses in its fluorescence intensity were observed after irradiation. Some of the photochemically active amino acids had lower than expected responses in amino acid sequencing experiments. This suggested specific sites of photochemical activity in the various peptides. The evidence for peptide crosslinks is also discussed.

Acrylamide quenching of tryptophan photochemistry and photophysics.

Studies of acrylamide quenching of tryptophan (Trp) fluorescence, photochemistry, and photoionization have been conducted. Quenching of Trp fluorescence in aqueous solution by addition of acrylamide in the concentration range 0.0-0.5 M was measured and resulted in a Stern-Volmer quenching constant of KSV = 21 +/- 3 M-1. Photolysis experiments were performed in which Trp was photolyzed at 295 nm in the presence of varying concentrations of acrylamide. The loss of Trp was monitored using reverse-phase high performance liquid chromatography (RP-HPLC) and was observed to follow first order kinetics. Production of N-formylkynurenine (NFK) was observed by RP-HPLC in irradiated Trp samples both in the presence and absence of added acrylamide. In addition, no new photochemical product was detected. This was taken as evidence that acrylamide did not alter the photochemical pathway but just reduced the reaction rate as expected for a physical quenching mechanism. Plotting the reciprocal of photolysis rate constant versus acrylamide concentration produced a Stern-Volmer constant for quenching of Trp photochemistry of KSV = 6 +/- 2 M-1. The KSV values for both fluorescence quenching and photolysis quenching were thus large, implying efficient quenching of both processes by acrylamide. Assuming an excited singlet state lifetime of 2.8 ns, the calculated second-order quenching rate constants for fluorescence and photolysis were kq = 7.5 x 10(9) and 2.1 x 10(9) M-1 s-1 respectively. The possible involvement of photoionization in the photolysis mechanism was investigated by studies of acrylamide quenching of voltage transients produced by xenon flash lamp excitation of Trp at aqueous/teflon or aqueous/mica interfaces.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluorescence quenching studies of the structures of calf gamma-II, III, and IV crystallins.

The structures of the calf lens crystallin fractions gamma-II, gamma-III, and gamma-IV have been investigated using the fluorescence quenching method. The three crystallin fractions showed very large differences in the quenching rates of their fluorescent tryptophan residues, for quenching by acrylamide or iodide in pH 7.5 phosphate buffer solutions. The experimentally measured quenching rate constants were kq(II) = 3.2 x 10(8), kq(III) = 9.9 x 10(8), and kq(IV) = 1.8 x 10(9) M-1 sec-1. Smaller rate constants were obtained for iodide quenching of the three crystallins, but the values were in approximately the same ratios as the ones found for acrylamide quenching. The conclusion is that the tryptophan residues in gamma-II crystallin are 6-10 times less easily quenched than those of gamma-IV crystallin and 3-6 times less easily quenched than those of gamma-III. These conclusions are in accord with those reached by Mandal et. al. based on fluorescence and CD data, who found the following order of Trp hydrophobicities: gamma-II greater than gamma-III greater than gamma-IV. The significance of these structural differences for lens function and stability remains to be elucidated.

Acrylamide and iodide fluorescence quenching as a structural probe of tryptophan microenvironment in bovine lens crystallins.

Fluorescence quenching using acrylamide and iodide quenchers has been used to investigate the microenvironments of tryptophan residues in bovine alpha-, beta-, and gamma-crystallin fractions. Acrylamide quenching is very sensitive to the degree of tryptophan accessibility to the solvent containing the acrylamide. Since acrylamide is able to diffuse into the interior of the protein, accessibility to acrylamide may result from Trp residues lying at the surface of the protein or from the existence of channels leading to the interior of the protein. Iodide ion is hydrated and is limited by its large size and charge to quenching of tryptophan residues lying at or near the surface of proteins. Tryptophan residues in the lens crystallin fractions were found to be highly accessible to acrylamide, yet the rate of quenching by acrylamide was very low, indicating that the tryptophan residues of the lens crystallin fractions occupy predominately hydrophobic environments. The high accessibility to acrylamide likely results from diffusion of acrylamide into the interior of the protein. Accessibility to iodide was much lower, as was the rate of quenching by iodide, adding support to the conclusions from acrylamide quenching.

Cataracts and photochemical damage in the lens.

The possibility is considered that ultraviolet radiation, from sunlight and other ambient sources, is a major causative factor in certain human cataracts. Epidemiological and experimental data from the literature are reviewed and new experimental results on exposure of rat lenses to UV laser radiation in vitro are presented. This UV irradiation of lenses is shown to cause: (i) photochemical alteration of tryptophan residues in lens proteins, resulting in loss of tryptophan fluorescence, (ii) new ultraviolet absorption bands attributable to photo-oxidation products of lens proteins, and (iii) cross-linking of polypeptides in lens proteins, detected by sodium dodecylsulphate-polyacrylamide gel electrophoresis. It is noted that the UV radiation-induced lens changes reported are quite similar to the changes found when normal human lenses develop cataracts. In order to establish the degree to which UV-exposed lenses resemble naturally occurring cataracts, a comprehensive comparison of anatomical, histological and biochemical parameters for normal, cataractous and UV-exposed human lenses is proposed.

Tyrosine-to-tryptophan energy transfer and the structure of calf gamma-II crystallin.

The efficiency of electronic energy transfer from tyrosine to tryptophan residues in the lens protein calf gamma II crystallin has been determined from measurements of fluorescence excitation spectra at 25 degrees C and independently from phosphorescence spectroscopy at 77 K. The total transfer efficiency from the fifteen tyrosines to the four tryptophans in native calf gamma II was found to be t = 78 +/- 10%, at 25 degrees C. The expected value based on Förster theory and the X-ray structure of the protein is t = 83%, in good agreement with the present experimental result. The transfer efficiency measured in denatured calf gamma II at 25 degrees C was t = 20 +/- 10%, in good agreement with the value t = 25% expected for the completely denatured protein, based on Förster theory and the known amino acid sequence. These results are of interest for several reasons. First, energy transfer can provide a simple , experimental confirmation of lens protein structures determined from X-ray data or from computer modeling studies. Second, the present studies show that energy transfer measurements can be used to monitor the effect of denaturants on lens protein structure, an aspect not readily investigated by X-ray crystallography. Third, the present electronic energy transfer studies may be relevant to understanding the mechanism of UV photodamage in lens proteins and hence in whole lenses.

The non-fluorescence of 4-fluorotryptophan.

The derivative 4-fluorotryptophan was confirmed to have negligible fluorescence at 25 degrees C and 285 nm (tryptophan/4-fluorotryptophan quantum-yield ratio greater than 100:1). However, photolysis experiments on tryptophan and 4-fluorotryptophan, in which loss of starting material was measured by reverse-phase h.p.l.c., demonstrated that 4-fluorotryptophan was significantly more photochemically active than the parent tryptophan, with the 4-fluorotryptophan photolysis quantum yield being 7 times larger than that of tryptophan at 25 degrees C and 285 nm. In addition, at 77 K and 275 nm 4-fluorotryptophan displayed strong fluorescence and phosphorescence, with emission quantum yields comparable with those of tryptophan at 77 K and 275 nm.

Light scattering and photocrosslinking in the calf lens crystallins gamma-II, III and IV.

The calf lens proteins gamma-II, -III and -IV crystallin have been photolyzed in pH 7.5 phosphate buffer solution at 25 degrees C. The photolysis light source was either a xenon arc lamp/monochromator system set to pass 290 +/- 5 nm or a nitrogen laser operating at 337.1 nm. Photolysis experiments at 337.1 nm were done both in the presence and absence of added 1.0 x 10(-4) M N-formylkynurenine (NFK). In addition, 1 x 10(-5) M riboflavin was added as a photosensitizer in a few of the experiments. All solutions were 1.0 mg ml-1 protein, and 1.0 ml of solution was irradiated for periods ranging from 10 min to 3 hr. During the 337.1 nm irradiations, the turbidity of the protein solutions was continuously monitored using a He-Ne laser at 632.8 nm. Progress of the 290 nm irradiations was monitored by observing the loss of tryptophan fluorescence for each of the gamma crystallin proteins. The rate of growth of light scattering, upon 337.1 nm irradiation, was greatest for gamma-IV. Addition of NFK caused the rates of growth of UV-induced light scattering of all three gamma crystallins to increase significantly. These rates were in the order: gamma-IV much greater than gamma-III greater than gamma-II. Following UV exposure, the protein solutions were analyzed using UV-visible absorption spectroscopy and SDS-PAGE. Irradiated gamma crystallin solutions showed increased optical density throughout the visible region, resulting from solution turbidity.(ABSTRACT TRUNCATED AT 250 WORDS)