Visible light neutralizes the effect produced by ultraviolet radiation in proteins.

The damage produced by UV-C radiation (100-280nm) in organisms and cells is a well known fact. The main reactions of proteins to UV-C radiation consist in the alteration of their secondary structures, exposure of hydrophobic residues, unfolding and aggregation. Furthermore, it has been found that electromagnetic radiation of lower energy (visible light, where wavelengths are between 400 and 750nm) also induces different disturbances in biomolecules. For instance, it has been observed that blue visible light from emitting diodes (LEDs) produces severe damage in murine cone photoreceptor-derived cells, and it can be even more harmful for some organisms than UV radiation. Recently, it has been found that the exposure of proteins to green and red light produces conformational changes, considerably increasing their cohesion enthalpies. This is presumably due to the strengthening of the hydrogen bonds and the formation of new ones. Therefore, it seems that visible light acts contrary to what it is observed for UV-C: instead of unfolding the proteins it folds them further, halting the damage produced by UV-C. This can be understood if we consider the modification of the folding energy-landscape; visible light induces the descent of the proteins into deeper states impeding the unfolding produced by UV-C.

Preventive role of lens antioxidant defense mechanism against riboflavin-mediated sunlight damaging of lens crystallins.

The main components of sunlight reaching the eye lens are UVA and visible light exerting their photo-damaging effects indirectly by the aid of endogenous photosensitizer molecules such as riboflavin (RF). In this study, lens proteins solutions were incubated with RF and exposed to the sunlight. Then, gel mobility shift analysis and different spectroscopic assessments were applied to examine the structural damaging effects of solar radiation on these proteins. Exposure of lens proteins to direct sunlight, in the presence of RF, leads to marked structural crosslinking, oligomerization and proteolytic instability. These structural damages were also accompanied with reduction in the emission fluorescence of Trp and Tyr and appearance of a new absorption peak between 300 and 400nm which can be related to formation of new chromophores. Also, photo-oxidation of lens crystallins increases their oligomeric size distribution as examined by dynamic light scattering analysis. The above mentioned structural insults, as potential sources of sunlight-induced senile cataract and blindness, were significantly attenuated in the presence of ascorbic acid and glutathione which are two important components of lens antioxidant defense system. Therefore, the powerful antioxidant defense mechanism of eye lens is an important barrier against molecular photo-damaging effects of solar radiations during the life span.

[Experimental study of influence of different damaging factors on lens. Report 3. Changes of lens protein composition].

Using differential electrophoresis protein composition of lens major proteins in hybrid mice F1 (C57B1XCBA) with cataracts of different etiology (senile, ultraviolet, radioactive and combined ultraviolet-radioactive exposure) was studied Changes that may be specific for cataract caused by aging, ultraviolet and/or gamma-irradiation were not revealed in water-soluble and water-insoluble protein fractions.

Simultaneous chemical and photochemical protein crosslinking induced by irradiation of eye lens proteins in the presence of ascorbate: the photosensitizing role of an UVA-visible-absorbing decomposition product of vitamin C.

Exposure to light has been implicated as a risk factor during aging of the eye lens and in cataract generation. In order to visualize the actual effect of UVA-visible light on this tissue, we incubated water-soluble eye lens proteins with ascorbate in the presence and absence of UVA-visible light for 3, 6 and 9 days at low oxygen concentration. The samples incubated in the presence of light were characterized by an initially small but continuous increase over time of the protein crosslinking. This was not the result of more extensive glycation because the decrease in amino group content of the proteins and the decomposition of ascorbate was the same in both irradiated and unirradiated samples. The augmented crosslinking capacity observed in the presence of UVA-visible light is due to the generation of a chromophore from the decomposition of ascorbate. This chromophore, obtained after 3, 6 and 9 days of incubation of solutions containing only ascorbate, induces both protein-crosslinking and oxidation after exposure to UVA-visible light in the presence of lens proteins. The extent of the crosslinking was proportional to the amount of the chromophore present in the solution. The presence of this chromophore was also determined when ascorbate was incubated with four-fold higher concentrations of N-α-acetyl lysine and N-α-acetyl arginine. When these samples were used as photosensitizers, the crosslinking degree was conditioned by the presence of this chromophore; nonetheless, the ascorbate-mediated advanced glycation end product (AGE) generation also made a contribution. The results of this work indicate that ascorbate oxidation, which generates the AGEs responsible for the chemical crosslinking of the lens proteins, also simultaneously produces a chromophore that can act as a photosensitizer, further increasing the protein crosslinking.

Mechanism of the very efficient quenching of tryptophan fluorescence in human gamma D- and gamma S-crystallins: the gamma-crystallin fold may have evolved to protect tryptophan residues from ultraviolet photodamage.

Proteins exposed to UV radiation are subject to irreversible photodamage through covalent modification of tryptophans (Trps) and other UV-absorbing amino acids. Crystallins, the major protein components of the vertebrate eye lens that maintain lens transparency, are exposed to ambient UV radiation throughout life. The duplicated beta-sheet Greek key domains of beta- and gamma-crystallins in humans and all other vertebrates each have two conserved buried Trps. Experiments and computation showed that the fluorescence of these Trps in human gammaD-crystallin is very efficiently quenched in the native state by electrostatically enabled electron transfer to a backbone amide [Chen et al. (2006) Biochemistry 45, 11552-11563]. This dispersal of the excited state energy would be expected to minimize protein damage from covalent scission of the excited Trp ring. We report here both experiments and computation showing that the same fast electron transfer mechanism is operating in a different crystallin, human gammaS-crystallin. Examination of solved structures of other crystallins reveals that the Trp conformation, as well as favorably oriented bound waters, and the proximity of the backbone carbonyl oxygen of the n - 3 residues before the quenched Trps (residue n), are conserved in most crystallins. These results indicate that fast charge transfer quenching is an evolved property of this protein fold, probably protecting it from UV-induced photodamage. This UV resistance may have contributed to the selection of the Greek key fold as the major lens protein in all vertebrates.

Tryptophan metabolites from young human lenses and the photooxidation of ascorbic acid by UVA light.

PURPOSE: To determine whether there are UVA light-responsive sensitizers in young human lenses capable of initiating the oxidation of ascorbic acid in the absence of oxygen. METHODS: Lens homogenates were fractionated, and low-molecular-weight (LMW) components were separated from the proteins by filtration through a 3000-MWt cutoff filter. Aliquots of each fraction were assayed for sensitizer activity by UVA irradiation (337-nm cutoff filter) with 0.1 mM ascorbic acid, measuring ascorbate oxidation by loss of absorbance at 265 nm. Two major peaks were isolated from a human lens water-soluble (WS)-LMW fraction on a reversed-phase column and were identified by mass spectrometry. RESULTS: All human lens fractions oxidized ascorbate when irradiated by UVA light. Most of the sensitizer activity in young human lenses was in the LMW fractions. An action spectrum for the WS-LMW fraction from human lens showed activity throughout the UVA region. Assays with and without oxygen showed little or no difference in ascorbate oxidized, arguing for a direct transfer of an electron in a so-called type 1 reaction. A human lens WS-LMW fraction contained two major peaks of activity. The greater peak was identified as 3-hydroxykynurenine glucoside (3OHKG) by mass spectrometry and its absorption spectrum, whereas the lesser peak was identified as 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid glucoside (AHBG). The activities were 1.1 and 2.8 nmol of ascorbate oxidized in 30 minutes/nmol 3OHKG and AHBG, respectively. CONCLUSIONS: The filter compounds present in human lenses can absorb UVA light and cause the oxidation of ascorbic acid in the presence and absence of oxygen, possibly initiating the glycation of lens proteins.

Autosensitized oxidation of glycated bovine lens proteins irradiated with UVA-visible light at low oxygen concentration.

Advanced glycation endproducts (AGEs) are generated in lens proteins following ascorbic acid glycation. Autosensitized photo-processes induced by these AGEs exposed to UVA-visible light at low oxygen concentrations, such as those found in the eye lens, were studied in order to establish their photo-damaging potential. Ascorbic acid glycated bovine lens proteins, irradiated with UVA and/or visible light at 5% oxygen concentration showed an increase in the amount of carbonyl groups and an increase in the emission of chemiluminescence. Both are indicators of the occurrence of amino acid oxidation within the proteins. A good correlation was found between the chemiluminescence intensity and the extent of the glycation process. No modifications in the SDS-PAGE patterns were observed when 6-day glycated proteins were irradiated with UVA-visible light at low oxygen pressure, excluding the occurrence of photochemical crosslinking. When the same samples were derivatized with 2,4-dinitrophenylhydrazine and analyzed by Western blot, using polyclonal antibodies to the DNP moiety, an increase in the carbonyl content restricted to the AGE-crosslinked proteins fractions was observed. These results indicate that photo-oxidations are limited to the nearby environment of the sensitizers. On the contrary, when native lens proteins were irradiated in the presence of small molecular weight models of AGEs, prepared by incubating ascorbate with Nalpha-acetylated lysine and arginine, the occurrence of both photochemically induced crosslinking and oxidative processes were observed, emphasizing the importance of the localization of the sensitizer. Taken together, these results provide information with respect to the actual photosensitizing ability of endogenous AGEs to induce photo-oxidative processes in lens proteins.

[Chaperon-like anticataract agents, the antiaggregants of lens crystallin. Communication 4. Study of the effect of a mixture of di- and tetrapeptides on a prolonged rat model of UV-induced cataract].

There is a potential of therapeutic action on certain stages of caractogenesis, in particular on the aggregation of water-soluble proteins of cytoplasmic lens fiber cells, giving rise to insoluble protein complexes. The effect of a combined preparation (N-acetyl carnosine and D-patethine), acting by the chaperon-like mechanism, was studied in vivo on a prolonged rat model of UV-induced cataract. The use of the combined preparation consisting of a mixture of peptides of N-acetyl carnosine and D-patethine in a ratio of 1:1 as ocular instillations and intraperitoneal injections could slow down the development of UV-induced cataract in vivo. Pathomorphological studies suggest that the combined preparation has a protective effect on lens tissue when the rat model of UV-induced cataract is employed.

[Search for chaperon-like anticataract agents, the antiaggregants of lens crystallin. Communication 3. Possibilities of a follow-up of caractogenesis processes on a prolonged rat model of UV-induced cataract].

To study the mechanisms of action of new-generation anticataract drugs, it is necessary to have an accessible and adequate model of age-related cataract. A model of UV-induced cataract is pathogenetically closest to that of age-related cataract. A prolonged rat model of UV-induced cataract developing within 10 months is proposed; the clinical features of UV-induced cataract have been established at different stages of its development. A moderate homogeneous cloud-like lenticular opacity was observed at the end of the experiment; a less pronounced homogeneous opacity was seen in the anterior and posterior cortical layers. Cataract development was assessed by the appraisal method using the developed rat lenticular transparency scale, as well as by microdensitometry of biomicroscopic lenticular optical sections. Within the proposed model, the pathomorphological lenticular changes are largely similar to the histological pattern of age-related cataract.

Tryptophan-derived ultraviolet filter compounds covalently bound to lens proteins are photosensitizers of oxidative damage.

The human eye is chronically exposed to light of wavelengths >300 nm. In the young human lens, light of wavelength 300-400 nm is predominantly absorbed by the free Trp derivatives kynurenine (Kyn), 3-hydroxykynurenine (3OHKyn), and 3-hydroxykynurenine-O-beta-D-glucoside (3OHKynG). These ultraviolet (UV) filter compounds are poor photosensitizers. With age, the levels of the free UV filters in the lens decreases and those of protein-bound UV filters increases. The photochemical behavior of these protein-bound UV filters and their role in UV damage are poorly elucidated and are examined here. UVA illumination of protein-bound UV filters generated peroxides (principally H2O2) in a metabolite-, photolysis-time-, and wavelength-dependent manner. Unmodified proteins, free Trp metabolites, and Trp metabolites that do not bind to lens proteins gave low peroxide yields. Protein-bound 3OHKyn (principally at Cys residues) yielded more peroxide than comparable Kyn and 3OHKynG adducts. Studies using D2O and sodium azide implicated 1O2 as a key intermediate. Illumination of the protein-bound adducts also yielded protein-bound Tyr oxidation products (DOPA, di-tyrosine) and protein cross-links via alternative mechanisms. These data indicate that the covalent modification of lens proteins by Kyn derivatives yields photosensitizers that may enhance oxidation in older lenses and contribute to age-related nuclear cataract.

Increased sensitivity of amino-arm truncated betaA3-crystallin to UV-light-induced photoaggregation.

PURPOSE: Exposure to UV-B light (wavelength, 290-320 nm) is a well-documented risk factor for age-related cataracts. As the lens ages, beta-crystallins tend to undergo proteolytic cleavage of their terminal extensions. To delineate the effects of loss of terminal arms on beta-crystallin function, the sensitivity of purified recombinant wild-type (rbetaA3) to UV-irradiation induced aggregation was compared with that of betaA3-crystallin missing the N-terminal extension (rbetaA3tr). METHODS: Proteins were expressed in baculovirus-infected Sf9 cells and purified by chromatography. Purified protein solutions (pH 7.4) were reduced by using Tris (2-carboxyethyl) phosphine HCl and irradiated with a 308-nm excimer laser at physiologically relevant UV doses and wavelengths (308 nm), and light-scattering (633 nm) was measured. Irradiated crystallins were analyzed by matrix-assisted desorption ionization (MALDI) and tandem liquid chromatography/mass spectrometry (LC-MS/MS). RESULTS: UV-irradiation of both rbetaA3 and rbetaA3tr resulted in major loss of soluble protein, as shown by absorption at 280 nm, size-exclusion chromatography (SEC) and SDS-PAGE, with concomitant formation of insoluble aggregates producing light-scattering. Compared with wild-type rbetaA3, rbetaA3tr showed a significant tendency to begin scattering light at lower UV dose and had a higher aggregation rate with increasing UV exposure. Changes in irradiated crystallins include aggregation and cross-linking, photolysis, and oxidation of methionine and tryptophan residues. CONCLUSIONS: Loss of beta-crystallin terminal arms appears to increase their tendency to aggregate in response to UV irradiation, suggesting that this loss in the maturing lens may increase susceptibility to age-related cataract.

Ocular lens blue autofluorescence cannot be used as a measure of individual cumulative UVR exposure.

BACKGROUND/PURPOSE: The accumulation of fluorophores in the ocular lens with age might be caused by ultraviolet solar radiation (UVR) exposure, but evidence of a relation between individual cumulative UVR exposure and lens autofluorescence is lacking. Individually determined UVR exposure has never before been related to lens autofluorescence, and the aim of this study was to investigate if ocular lens blue autofluorescence can be used as a biological UVR dosimeter. METHODS: Ocular lens autofluorescence was quantified in vivo by fluorescence spectroscopy in 145 volunteers (108 healthy subjects, 18 with basal cell carcinoma (BCC) and 19 with cutaneous malignant melanoma (MM)). The excitation wavelength was 350 nm and the fluorescence emission was 450 nm. Individual UVR exposure data were collected both retrospectively and prospectively using questionnaires and electronic personal UVR dosimeters. RESULTS: Lens blue autofluorescence increased significantly with age (P=0.01), and females had significantly higher autofluorescence than males (P=0.024); the two factors explained 10% of the total variation in lens autofluorescence. Neither smoking habits nor use of glasses/contact lenses or sunglasses influenced autofluorescence. No correlations between autofluorescence and UVR exposure measurements were found, and neither was there a difference in autofluorescence between groups with high and low UVR exposure (P-values>0.1), respectively. MM patients had significantly (P=0.019) higher autofluorescence than healthy subjects when age and sex differences were taken into account; no such difference (P=0.097) was detected between BCC patients and healthy subjects. CONCLUSION: The results indicate that age and gender only play a minor role in the level of lens blue autofluorescence. Exposure to UVR has been suggested to be responsible for a part of the age-related increase in autofluorescence, but this could not be confirmed in this study. The higher level of lens autofluorescence found in MM patients might be due to genetics rather than higher cumulative UVR exposure. In conclusion, ocular lens blue autofluorescence cannot be used as a biological UVR dosimeter.

Minimization of photooxidative insult to calf lens protein irradiated with near UV-light in the presence of pigmented glucosides derived from human lens protein.

An aqueous solution of a pigmented glucoside associated with human lens protein, 2-amino-3-hydroxyacetophenone-O-beta-D-glucoside (AHA-Glc), was irradiated with near UV-light. The near UV-irradiated glucoside was shown to generate a much lower level of active species of molecular oxygen as compared to the level of the active species generated from the irradiated aglycon, 2-amino-3-hydroxyacetophenone (AHA). This result suggests that the glycon of the glucoside is functioning as a scavenger for active oxygen generated from the aglycon of the irradiated glucoside. Superoxide dismutase (SOD) was shown to remove a large portion of the active oxygen generated from the irradiated AHA, so the bulk of the active species generated is assumed to be superoxide anion. The small portion of active oxygen remains after removal of superoxide anion may include singlet oxygen. The photooxidation of tryptophan residues of calf alpha-crystallin irradiated with near UV-light in the presence of AHA-Glc or AHA was investigated to confirm the role that the glycon plays in diminution of the active species of oxygen generated through the photosensitized aglycon of the glucoside. A decrease with time in the fluorescence intensity of the tryptophan residues irradiated with AHA-Glc was shown to be much slower as compared to the time-dependent decrease with AHA, indicating that the photooxidation proceeds with an increase in accumulation of active oxygen generated through the aglycon and that the glycon of the glucoside deactivates the active species as it is formed in the photodynamic process. Similar effects have also been observed in calf lens crystallin irradiated with either 3-hydroxykynurenine-O-beta-D-glucoside (HKN-Glc) or 3-hydroxykynurenine (HKN). Furthermore, effects of near UV-irradiation on calf lens soluble protein in the presence of AHA-Glc or AHA were studied by monitoring changes in the SDS-PAGE profile of the irradiated protein. Near UV-irradiation with AHA-Glc was shown to bring about a slight change in cross-linking of the polypeptides, while irradiation with AHA was shown to give rise to a significant increase in cross-linking of the polypeptides. In conclusion, pigmented glucoside associated with human lens protein is not only a photosensitizer for near UV-light but also an anti-photooxidant to deactivate active oxygen formed through the in situ photosensitizer, in order that photooxidative insults to lens proteins may be minimized during aging.

The effect of UVA light on the anaerobic oxidation of ascorbic acid and the glycation of lens proteins.

PURPOSE: To determine whether UVA-excited human lens chromophores can cause the oxidation of ascorbic acid in the absence of oxygen, and whether these oxidation products are capable of glycating lens proteins. METHODS: The oxidation of ascorbic acid, mediated by UVA irradiation in the presence of aged human lens proteins, was measured in the absence of oxygen by the decrease in absorbance at 265 nm in vitro. An action spectrum from 320 to 400 nm was determined for both ascorbate oxidation and the photobleaching of the lens yellow pigments at lambda = 350 nm. The UVA-mediated oxidation products of [U-(14)C]ascorbate were quantified by HPLC. Glycation was assayed by the UVA-dependent incorporation of [U-(14)C]ascorbate into lens proteins with a water-insoluble (WI) fraction in vitro, with incubated whole human lenses, and with a WI fraction after a 5- to 7-day exposure to ambient sunlight. An enzymatic digest of [U-(14)C]ascorbate-labeled proteins was fractionated over HPLC columns and compared with the 330-nm absorbance profile of a proteolytic digest of aged human lens proteins. RESULTS: Aged human lens WI proteins absorbed UVA light (86 J/h per square centimeter) and oxidized 33 to 45 nanomoles of ascorbate over 1 hour in the absence of oxygen. No ascorbate oxidation was detected, however, in the dark control. An action spectrum showed that ascorbate oxidation occurred throughout the UVA region, with lambda(max) at 350 nm, which was similar to the action spectrum obtained for the photobleaching of the lens chromophores. Anaerobic UVA irradiation of aged human lens proteins for 2 hours with [U-(14)C]ascorbate resulted in a 40% loss of ascorbate with the accumulation of dehydroascorbic acid, diketogulonic acid, and oxalate. After subsequent incubation for 24 hours, the ascorbate oxidation products disappeared, with a corresponding incorporation of radioactivity into lens proteins. Chromatography of enzymatic digests of the labeled proteins produced peaks that coeluted with several of the 330-nm absorbing peaks in an aged human lens protein digest. Irradiation of whole human lenses for 2 hours caused a 33% loss of total lens ascorbate. UVA irradiation of aged human lenses for 2 hours resulted in the incorporation of ascorbate into lens proteins during the ensuing 24 hours in the dark. Exposure of aged human lens WI proteins to reflected ambient sunlight (1.1 J/h per square centimeter) for 5 to 7 days in the absence of oxygen also produced an increased incorporation of [(14)C]ascorbate into protein when compared with dark control samples. CONCLUSIONS: These data argue that UVA light can cause an oxidation of ascorbic acid in the absence of oxygen, due to the activation of the sensitizers present in aged human lens WI proteins. The oxidation products formed were the same as those seen in the presence of oxygen, and were rapidly incorporated into protein, apparently by Maillard-type chemistry. These data argue that ascorbate glycation can occur under the low oxygen levels thought to exist in the human lens nucleus in vivo.

Effect of long-term dietary manipulation on the aggregation of rat lens crystallins: role of alpha-crystallin chaperone function.

PURPOSE: To investigate the effect of food, protein, and vitamin restriction on the susceptibility of lens crystallins to aggregation and chaperone activity of alpha-crystallin. METHODS: Thirty day old Wistar/NIN rats were maintained on regular rodent diet (C), 50% food restriction (FR), 75% protein restriction (PR), and 50% vitamin restriction (VR) diet for 20 weeks. At the end, alpha-, beta-, and gamma-crystallins were isolated from the lenses of these animals and subjected to in vitro aggregation induced by oxidation, UV irradiation and heat. Aggregation and chaperone activity was assessed by light scattering methods. RESULTS: Dietary restriction has been shown to extend the mean and maximum life span and retard age-related diseases, including cataract. In this study, we demonstrate that while beta- and gamma-crystallins isolated from FR and PR groups were less susceptible to in vitro induced aggregation, beta- and gamma-crystallins from the VR group were more susceptible, compared to controls. Alpha-crystallin from any of the groups did not shown a considerable amount of aggregation. On the other hand, the chaperone activity of alpha-crystallin from FR and PR groups was not significantly different from controls. However, alpha-crystallin from the VR group demonstrated substantially higher chaperone activity than controls. CONCLUSIONS: These results indicate that while food and protein restriction appear to lower the susceptibility of beta- and gamma-crystallins towards aggregation, vitamin restriction tends to increase the aggregation. Chaperone activity of alpha-crystallin is affected (improved) by only vitamin restriction.

Comparison of ultraviolet induced photo-kinetics for lens-derived and recombinant beta-crystallins.

PURPOSE: The photobiology of purified recombinant crystallins has not been studied. Here we examine photo-induced aggregation of purified recombinant mouse betaA3-crystallin (rbetaA3) and compare it with that of betaL-crystallins isolated from bovine lenses. METHODS: rbetaA3-Crystallin was expressed in baculovirus-infected Sf9 cells and purified by ion-exchange and gel-filtration chromatography. Protein solutions (pH 7.4) were irradiated at room temperature using a 308 nm excimer laser and light scattering was registered by attenuation of an unabsorbed beam of red light (670 nm). RESULTS: Irradiation of bovine alpha-crystallin, betaL-crystallin, rbetaA3-crystallin and gammaB-crystallin resulted in formation of insoluble aggregates with subsequent light scattering. Different slopes and threshold energies were observed for light scattering by each of these species. Sensitivity to ultraviolet irradiation induced light scattering as determined from threshold energies varied, with gamma-crystallins showing the greatest sensitivity, the betaL- and rbetaA3-crystallins showing an intermediate sensitivity and alpha-crystallins much less sensitive. Low doses (100 J/cm2) resulted in irreversible formation of water soluble oligomers but no insoluble aggregates as indicated by changes in light transmission. The photo-behavior of rbA3 was similar to mixed betaL-crystallin and different from that of alpha- and gamma-crystallins. CONCLUSIONS: Ultraviolet induced sensitivity of purified recombinant crystallins reflects that of mixed crystallin populations and should provide an indication of the pathogenicity of specific crystallin sequence changes associated with lens aging and hereditary cataract.

Correlation between the loss of the chaperone-like activity and the oxidation, isomerization and racemization of gamma-irradiated alpha-crystallin.

Alpha-crystallin possesses a molecular chaperone-like activity that prevents proteins from aggregating; however, the mechanism of this activity is not well known. Here we have taken gamma-irradiated alpha-crystallin and studied the relationship between the decrease in chaperone-like activity and the modifications such as oxidation, isomerization and racemization of amino acids in this molecule. We found that the chaperone-like activity of alpha-crystallin decreased with increasing gamma irradiation. After 4000 Gy gamma irradiation the activity of alpha-crystallin was reduced to 40% of the level of nonirradiated, native alpha-crystallin. The circular dichroism spectrum showed that the secondary structure of the irradiated alpha-crystallin had not changed. However, its tertiary structure appeared to change following more than 1000 Gy irradiation. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis also indicated that cross-linking of alpha-crystallin increased with increasing radiation doses. Irradiated and nonirradiated alpha-crystallin was subjected to trypsin digestion and peptide analysis by reverse-phase high-performance liquid chromatography and mass and sequence analysis. Depending on the radiation dose, Met-1 of alpha A-crystallin was oxidized to methionine sulfoxide. In addition, Asp-151 of alpha A-crystallin was isomerized to the beta-Asp form after irradiation, and racemization of Asp-151 decreased. Thus, the loss of the chaperone-like activity of alpha-crystallin is related to changes in its isomerization, oxidation and racemization.

Spectroscopic study of visible-light effects on hypericin-lens proteins systems.

Molecular interactions between hypericin and alpha-, beta- and gamma-crystallin proteins have been studied by means of absorption and steady-state fluorescence spectroscopy, aiming to clarify if and how the pigment binds to the proteins and to investigate the effects of visible-light irradiation on these molecular systems. Such a study is a prerequisite for assessing the possibility of using hypericin as a mild antidepressant and/or as a photodynamic agent for the treatment of eye tumors and eye viral and bacterial diseases without side injuries to the lens. We have shown that in dark-kept samples, with increasing alpha-crystallin concentration, both the fluorescence emission intensity and the ratio of the absorption maxima around 590 and 550 nm of hypericin increase. These effects have been attributed to the monomerization of nonfluorescent hypericin aggregates caused by the binding of the pigment to alpha-crystallin. The binding constant of hypericin has been evaluated to be of the order of 3.0 (mg/mL)-1, corresponding to a dissociation constant of the order of 0.3 mg/mL. Following irradiation with light of wavelengths over 400 nm, at an irradiance of 20 mW/cm2, both tryptophan and hypericin fluorescence emission intensities decrease. These effects are suggested to be the consequence of a spatial rearrangement of the protein framework which takes place following the alpha-crystallin photopolymerization sensitized by hypericin itself described in the literature. For the sake of comparison hypericin has been studied also in the presence of beta H-, beta L- and gamma-crystallins at the same concentration.