CHANGES IN TERTIARY STRUCTURE OF CALF-LENS α-CRYSTALLIN BY NEAR-UV IRRADIATION: ROLE OF HYDROGEN PEROXIDE

The effect of 300 nm irradiation on the three lens crystallins, α-, β-, and γ-, was studied by using fluorescence and circular dichroism techniques. α-Crystallin showed a pronounced change in tertiary structure as manifested in fluorescence and circular dichroism measurements. This finding is in agreement with our earlier findings that the tryptophan residues of α-crystallin are more exposed than those of the other two crystallins. The results of studies using inhibitors specific for the different active species of oxygen suggest that H₂O₂-mediated damage is involved in the change of tertiary structure of the proteins. Analyses of circular dichroism spectra indicate that, upon irradiation, the secondary structure of α-crystallin remains virtually unaltered, and that the change in tertiary structure results primarily from photoinduced damage to the tryptophan residues.     

CONFORMATIONAL CHANGES OF BOVINE LENS CRYSTALLINS IN A PHOTODYNAMIC SYSTEM

Abstract— Conformational changes of bovine lens crystallins in a photodynamic system generating singlet oxygen, have been investigated. The formation of intersubunit crosslinks was observed in all three classes (α-, β and γ-) of crystallins by irradiation in the presence of the photosensitizer methylene blue. Near-UV circular dichroism (CD) spectra of the crystallins were significantly altered by irradiation under these conditions, indicating changes in tertiary structure but the far-UV CD remained unchanged suggesting that the secondary structure ((β-sheet conformation) remains unchanged. Significant changes in the absorption and fluorescence spectra were also observed. Measurement of total sulfhydryl content showed a decrease of 27%, 50% and 37% for α-, β- and γ-crystallins respectively, after irradiation. Fluorescence lifetime measurements of N-iodoacetyl-N'-(5-sulfo-l-naphthyl)ethylenediamine-labeled crystallins showed a significant decrease of the lifetime of the major decay components of the label bound to sulfhydryl groups of α- and γ-crystallins, but showed no change in the microenvironment of the sulfhydryl groups of β-crystallin. The results are consistent with the microenvironments of the tryptophan and sulfhydryl groups predicted from sequence studies.     

Radiolysis-Induced Oxidation of Bovine α-Crystallin

Abstract— Radiolysis of water by ionizing radiation results in the production of pure hydroxyl radicals. This technique, combined with analysis by tandem mass spectrometry (MS/MS), has been used to study the effect of hydroxyl radicals on the intact bovine α-crystallin protein. After exposure to -γ-irradiation, the oxidized α-crystallin was digested with trypsin and the resulting peptides were fractionated by reverse-phase HPLC. The isolated fractions were analyzed by matrix-assisted laser desorption ionization and by MS/MS to determine the locations and identities of the modifications. Structural analysis revealed that methionine 1 of αA- and αB-crystallin and methionine 68 of αB-crystallin were oxidized to methionine sulfoxide. Hydroxytryptophan was formed from each tryptophan residue in α-crystallin, although only tryptophan 9 of αA-crystallin was converted into N-for-mylkynurenine. This study has, for the first time, identified the sites of modification and the structures produced in the intact α-crystallin protein by exposure to hydroxyl radicals. By determining the consequences of in vitro exposure of α-crystallin to pure hydroxyl radicals, the in vivo contribution of this reactive oxygen species to the overall oxidative stress of the lens will be achieved from the identification of the modifications to α-crystallin purified from intact human lenses.     

NEW PHOTO-CONVERTIBLE REACTIONS OF BLUE-FLUORESCENT CALF α-CRYSTALLIN

Abstract— Both native blue fluorescent α-crystallin from calf lenses and UV (300 nm)-irradiated blue-fluorescent α-crystallin, when further irradiated with 365 nm-UV light, produce photo-products capable of emitting a new fluorescence at 455 nm. Illumination of the photo-products with 420 nm visible light regenerates the original fluorescence at 420–425 nm. In addition, another fluorescence at 400 nm has also been found in UV (300 nm)-irradiated blue-fluorescent α-crystallin, when exposed to 365 nm-UV light.     

Photosensitized Effects of Rose Bengal on Structure and Function of Lens Protein "Alpha-Crystallin"

The conformational changes of the bovine lens protein "α-crystallin" have been investigated in the presence of the photosensitizer Rose Bengal (RB), in the dark as well as after visible light irradiation. Absorption and fluorescence emission spectra of RB [5 × 10⁻⁶  m ] and Fourier transform-IR spectra of α-crystallin [5 mg mL⁻¹] were significantly altered upon RB α-crystallin complex formation. RB was found to bind to α-crystallin in a molecular pocket characterized by a low polarity, with Trp most likely involved in this interaction. The binding constant ( K _b) has been estimated to be of the order of 2.5 (mg/mL)⁻¹. The intrinsic fluorescence of α-crystallin was quenched through both dynamic and static mechanisms. Light-induced photosensitized effects showed structural modifications in α-crystallin, including tertiary and secondary structure (an increase in unordered structure) alterations. Notwithstanding those photoinduced structural variations detected in α-crystallin when complexed with RB, the protein still retains its ability to play the role of chaperone for β-crystallin.     

Identification of Photooxidation Sites in Bovine α-Crystallin

Abstract— Because UV irradiation of proteins can produce reactive oxygen species and exposure to UV light has been implicated in cataractogenesis, the sites of photooxidation of bovine α-crystallin, a major lens protein with molecular chaperone activity, were identified using tandem mass spectrometry (MS/MS). Bovine α-crystallin was irradiated with UV light (293 nm) for 1, 4 and 8 h, digested with trypsin and analyzed by matrix-assisted laser de-sorption ionization, time-of-flight mass spectrometry (MALDI) to identify the oxidized sequences. Tryptic peptides were purified by reverse-phase HPLC and oxidized peptides were sequenced by MS/MS to determine the sites of oxidation. Tryptophan fluorescence decreased exponentially with increasing time of UV exposure and peptides containing residues 1-11 of α-crystallin and 1-11, 12-22 and 57-69 of α-crystallin were determined to be oxidized by shifts of 16 D or multiples of 16 Da above the mass of the unmodified peptide. The MALDI analysis revealed single oxidation of all four sequences, which increased with increasing time of UV exposure and possible double oxidation of α 12-22. The specific sites of photooxidation indicate that the N-terminal regions of α-and β-crystallin are exposed to an aqueous environment and are in the vicinity of tryptophan residues from neighboring subunits.     

THE EFFECTS OF NEAR-UV RADIATION ON HUMAN LENS β-CRYSTALLINS: PROTEIN STRUCTURAL CHANGES and THE PRODUCTION OF O2_ and H2O2

beta-Crystallins (beta 1-, beta 2- and beta 3-crystallin) comprise nearly half the protein of the human lens. The effect of near-UV radiation, which is one of the possible risk factors in cataract formation, on the beta-crystallins is investigated in this study. Protein intersubunit crosslinking, change in charge of the protein subunits to more acidic species and changes in protein tertiary structure (conformation) by 300 nm irradiation are reported. The fluorescence yield of protein tryptophan residues decreases by 300 nm irradiation. There is an increase in nontryptophan fluorescence (lambda cx 340 nm, lambda cm 400-600 nm), and in protein absorption at 340 nm, due to the formation of tryptophan photooxidation products. Both tryptophan and its oxidation products can be photoexcited by 300 nm irradiation and the latter are known to be good photosensitizers. The results provide evidence for the generation of H2O2 in the irradiated human beta-crystallin solutions by the Type I photosensitizing action of the chromophores absorbing at 300 nm. The H2O2 is generated via the intermediate production of O2 anion; the latter spontaneously dismutates to H2O2, presumably via O2- protein interactions. The amount of H2O2 generated per absorbed photon is compared for various solutions of beta 1-, beta 2- and beta 3-crystallins from human lenses of different age.     

CHANGE IN SULFHYDRYL GROUP MICROENVIRONMENT OF CALF LENS α-CRYSTALLIN BY 300 nm LIGHT

Abstract— The effect of 300 nm irradiation on the sulfhydryl groups of calf lens a-crystallin has been investigated by using specific, covalently bound fluorescent sulfhydryl probes 4–(N-iodoacetoxy)ethyl-N-methylamino-7-n-itrobenz-2-o-xa-1,3-d-iazole (IANBD), N-iodoacetyl-N'-(5-s-ulfo-l-naphthyl) ethylene-diamine (1,5 IAEDANS) and 5-i-odoacetamidofluorescein (IAF). The decrease in tryptophan fluorescence with time of irradiation of a-crystallin, is accompanied by a decrease in the fluorescence of the hydrophobic sulfhydryl label IANBD. In addition, the fluorescence of the surface-sulfhydryl label IAF increased in the irradiated a-crystallin. These results indicate that the sulfhydryl groups are in a more exposed (hydrophilic) environment in the irradiated protein than in the control, possibly because of partial unfolding of the protein. This result is confirmed by fluorescence lifetime measurements with IAEDANS. The decay curve of IAEDANS-α-crystallin has a major lifetime of 15.7 ns and a minor one of 24.6 ns. Upon irradiation, the lifetime of the major component decreases to 10.2 ns and that of the minor component to 21.7 ns. Denatured IAEDANS-α-crystallin has a single lifetime of 10.4 ns. These results show that the photoinduced damage to the tryptophan residues of α-crystallin alters the environment of the sulfhydryl groups and induces a change in the tertiary structure of the protein. Proximity of the cysteine residues to tryptophan in the tertiary structure of the protein may be an important determinant of their susceptibility to photoinduced change.     

Oxidation-Induced Structural Alterations and Its Effect on Chaperone Function of Rat Lens α-Crystallin

An ascorbate-FeCl₃-EDTA-H₂O₂ system was used to oxidize rat lens α-crystallins. Under this oxidative insult, the chaperone activity of α-crystallin toward γ-crystallin was shown to decrease significantly, which is quite different from the result reported by Wang and Spector. (Invest. Ophthalmol. Vis. Sci. 1995 , 36, 311-321.) Fluorescence spectroscopy and circular dichroism were employed to characterize the structural changes of oxidized α-crystallin. It was found that fluorescence intensity of l-anilinonaphthalene-8-sul-phonate (ANS) bound to oxidized α-crystallin increased comparing to that bound to normal α-crystallin, suggesting oxidation causes the exposure of more hydrophobic regions. Further, α-crystallin's fluorescence intensity in response to tryptophan residues showed a pseudo first order decline. Amino acid analysis of normal versus oxidized α-crystallin confirmed actual decline in tryptophan levels, showing about 80% of tryptophan being modified after 10-hour oxidation. Circular dichroism showed both changes in the secondary and tertiary structures of oxidized α-crystallin, characterized by a large loss of aromatic-type amino acid interactions and a large loss of β-sheet structure. In conclusion, modified tryptophan, secondary and tertiary structural changes of α-crystallin correlate best with the reduction of chaperone function, the curves all showing a linear slope for 10 hours, then plateauing. These results indicate that the decrease of α-crystallin chaperone activity is attributed to the structural changes.     

Fluorescence Study on the Interaction Between Hypericin and Lens Protein "α-Crystallin"

Hypericin has been reported as a potent photosensitizing agent exhibiting antiviral, antibacterial, antineoplastic activities. Although its photophysics and mode of action are strongly modulated by the binding protein, detailed information about its mechanism of interaction with possible cellular targets, including proteins, is still lacking. Previous in vitro studies demonstrated that hypericin can be uptaken by intact lens and is able to bind to the major lens protein "α-crystallin." In this study, the mechanism of interaction of this potent drug with α-crystallin was studied using the chemical denaturant guanidine hydrochloride (GdnHCl) and the hydrophobic surface probe, 8-anilino-1-naphthalenesulfonic acid (ANS). Fluorescence measurements showed that the increased exposure of tryptophan resulting from partial unfolding of α-crystallin incubated with 1.0 mol L⁻¹ of GdnHCl corresponds to the maximum accessibility of hydrophobic sites to ANS at the same GdnHCl concentration. Interestingly at this additional hydrophobicity of the protein, hypericin exhibited its maximum fluorescence intensity. This in vitro study implied that hydrophobic sites of α-crystallin play a significant role in its interaction with hypericin. The binding between α-crystallin and hypericin was found to be enhanced by partial perturbation of the protein.     

CONFORMATIONAL ANALYSIS OF THE RHODOPSIN CHROMOPHORE USING BICYCLIC RETINAL ANALOGUES

Abstract— For investigation of the chromophore conformation around the trimethyl cyclohexene ring and of the origin of the induced β-circular dichroism band in rhodopsin, two C₆-C₇ single bond-fixed retinal analogues, 6s-cb- and 6s-trans-locked bicyclic retinals (6 and 7, respectively) were synthesized and incorporated into bovine opsin in CHAPS-PC mixture. 6s-cb- and 6s-tram-Locked rhodopsin analogues (8 and 9 ) with A max at 539 and 545 nm, respectively, were formed. Interestingly, both 8 and 9 displayed α- and β-circular dichroism bands. The ellipticity of α-bands are similar in each other, while the β-band of 8 was about three times stronger than that of 9. Irradiation of 6s-trans-locked rhodopsin, 9, in the presence of hyroxylamine, resulted in the formation of only one of the enantiomers of 6s-rrans-locked retinal oxime showing a positive circular dichroism signal at around 390 nm. This fact strongly suggests that the retinal binding site of rhodopsin shows a chiral discrimination. From these experimental results, the interactions between the trimethyl cyclohexene ring portion in the chromophore and the neighbouring protein moiety in the rhodopsin molecule are discussed.     

NEAR UV LIGHT AND CATARACTS*

Abstract. Sunlight and many types of artificial lighting contain near-UV light (300–400 nm). These wavelengths can enter the eye and are maximally absorbed in the lens due to its chemical composition. The lenses of certain animals develop cataracts from exposure to this light, and changes similar to those that occur in human lenses with certain types of cataracts and with aging are inducible in isolated human lenses. These changes seem to be associated with chemical alterations in the essential amino acid tryptophan either as a part of proteins or in free form. Such changes in tryptophan would result in lens cell toxicity, in increased pigmentation of the lens, and in large aggregates of proteins. The latter two changes would result in losses in the ability of the lens to transmit visible light needed for vision, and the abnormal state called cataract would result. Much more work is needed to prove that near-UV light can accelerate cataractous changes in the lenses of living humans. Studies at the basic chemical level are needed, but population studies would be most essential for the final proof. Many preventive measures could become available, including the use of special types of spectacles and dietary additives.     

STUDIES ON THE PROTEIN CONFORMATION OF PHYTOCHROME

Abstract— The extinction coefficients for large rye phytochrome were found to be: Fluorescence and circular dichroism spectra of large- and small-molecular-weight rye phytochrome give no evidence for a protein conformational change on phototransformation of phytochrome. The large molecule has a fluorescence emission peak at 331 nm when excited at 290 nm, and an excitation peak for this emission at 288 nm. The circular dichroism spectra indicate that large rye phytochrome has about 17–20% a-helix content, 30%β-structure and 50% random coil, and that the small rye phytochrome has about 10–13%α-helix content. The ultraviolet difference spectra for large and small rye phytochrome are similar and differ from the difference spectrum of the small oat phytochrome in the relative size of the 296–298 nm peak. The difference spectra may reflect changes in chromophore absorbance and in the environment of amino acid residues near the chromophore, particularly of tyrosine, and perhaps of tryptophan and cysteine.     

ASCORBIC ACID GLYCATION OF LENS PROTEINS PRODUCES UVA SENSITIZERS SIMILAR TO THOSE IN HUMAN LENS

-Soluble calf lens proteins were extensively glycated during a 4 week incubation with ascorbic acid in the presence of oxygen. Amino acid analysis of the dialyzed proteins removed at weekly intervals showed an increasing loss of lysine, arginine and histidine, consistent with the extensive protein cross-linking observed. Irradiation of the dialyzed samples with UVA light (1.0 kJ/cm² total illumination through a 338 nm cutoff filter) caused an increasing loss of tryptophan, an additional loss of histidine and the production of micromolar concentrations of hydrogen peroxide. No alteration in amino acid content and no photolytic effects were seen in proteins incubated without ascorbic acid or in proteins incubated with glucose for 4 weeks. The rate of hydrogen peroxide formation was linear with each glycated sample with a maximum production of 25 nmol/mg protein illuminated. The possibility that the sensitizer activity was due to an ascorbate-induced oxidation of tryptophan was eliminated by the presence of a heavy metal ion chelator during the incubation and by showing equivalent effects with ascorbate-incubated ribonuclease A, which is devoid of tryptophan. The ascorbate-incubated samples displayed increasing absorbance at wavelengths above 300 nm and increasing fluorescence (340/430) as glycation proceeded. The spectra of the 4 week glycated proteins were identical to those obtained with a solubilized water-insoluble fraction from human lens, which is known to have UVA sensitizer activity. The incubation of lens proteins with dehydroascorbic acid or l -threose, but not fructose, produced equivalent glycation, protein crosslinking and sensitizer activity. The relative sensitizer activity of the 4 week glycated sample was quantitatively very similar to that of a water-insoluble fraction from aged human lenses. These data are consistent with the hypothesis that the protein-bound brunescence in the lens may be advanced glycation endproducts, which are formed in large part by the oxidation products of ascorbic acid, and that these compounds may contribute significantly to the UVA sensitizer activity present in aged human lenses.     

ULTRAVIOLET ACTION SPECTRUM FOR FLUOROGEN PRODUCTION IN THE OCULAR LENS

Abstract— …Previous work has demonstrated that fluorescent material (360nm excitation, 440nm emission), whose concentration normally increases with age in human lenses, can be generated artificially by exposing cultured human or animal lenses to UV radiation. In the present paper we report measurements of the rate of production of this fluorescent material in rat lenses in vitro as a function of UV irradiation wavelength. A plot of the observed rate of fluorogen production normalized to constant photon flux vs irradiation wavelength shows little action at 360 or 320nm, increases sharply at 300nm, remains relatively constant in the range 300–280nm, and then exhibits a further gradual rise from 270–250nm. The results on rat lenses are compared with results reported elsewhere for tryptophan in aqueous solution. The action spectrum for photochemical destruction of tryptophan in solution closely parallels that for fluorogen production in rat lenses. This result and other evidence suggest that photochemical destruction of tryptophan might be the initial event in UV-induced fluorogen production in the ocular lens.     

STRUCTURE AND STABILITY OF γ-CRYSTALLINS-IV. AGGREGATION AND STRUCTURAL DESTABILIZATION IN PHOTOSENSITIZED REACTIONS

Abstract— Unlike α- and β-, γ-crystallins become turbid upon irradiation with 300 nm or white light in the presence of photosensitizers, e.g., methylene blue (MB) or riboflavin (RF). These proteins, however, do not aggregate in the presence of guanidine hydrochloride. Turbidity formation is concentration-dependent. Except in RF-sensitized reactions, the onset and rate of turbidity formation are faster in γ-IV than in the other two crystallins. Labeling the thiol groups of the protein with iodoacetamide does not change the turbidity rate in case of irradiation with 300 nm light, but it does change it in case of RF- or MB-sensitized reaction. The order of rate constants of the decrease in tryptophan emission under aerobic conditions upon 300 nm irradiation and MB- and RF-sensitized reactions is γ-IV > γ-III > γ-II. The rate constants in the absence of air and in the presence of D₂O upon MB- and RF-sensitized reactions also indicate that the role of singlet oxygen is significant in the former reaction. We suggest that the photoinduced structural changes occur in two steps. In the first, photooxidation of tryptophan as well as cysteine residues (including the buried cysteines) occurs, leading to small conformational changes of the protein. Conformational changes of the protein, as evident from the near-UV CD and fluorescence lifetime measurements, subsequently result in aggregation of the protein. As suggested by X-ray analysis, the perturbation of the cys 78 and 32 by the active species of oxygen appears to be responsible for the destabilization of the protein structure, resulting in rapid aggregation of these crystallins.     

ALTERATION OF DYNAMIC QUATERNARY STRUCTURE AND CALCIUM-BINDING ABILITY OF ß-CRYSTALLIN BY LIGHT

Abstract— ß-crystallin, one of the three main constituent proteins of the eye lens, exists as an equilibrium population of oligomeric (ßH), trimeric (ßL1) and dimeric (ßL2) species. This equlibrium is dependent on various factors such as the protein concentration, ionic strength and pH of the medium. WE have studied the effect of ultraviolet B radiation on the aggregational patterns of ß-crystallin, using size-exclusion chromatography. Irradiation of a solution of ßH-crystallin at 295 nm for about 30 min causes the deaggregation of the hexameric population into dimers. Irradiation for a longer time, however, produces cross-linked high molecular weight products. Irradiation of a ßL2 solution for 30 min does not perturb the elution profile, while irradiation for a longer time increases the content of ßL1 (trimeric) crystallin. Irradiation also causes a decrease in the calcium-binding affinity of the ß-crystallins.     

Photooxidation of lens proteins with xanthurenic acid: a putative chromophore for cataractogenesis.

The tryptophan metabolite, xanthurenic acid (Xan), is produced through a transamination reaction in high concentrations in human lenses with age and has been isolated from aged human cataractous lenses. It has appreciable absorption between 300 and 400 nm (lambda max = 334 nm), the range absorbed by the human lens. Our recent studies have shown that unlike most tryptophan metabolites in the eye, Xan is photochemically active, producing both superoxide and singlet oxygen. To determine if Xan could act as a photosensitizer and photooxidize cytosolic lens proteins, alpha-, beta- and gamma-crystallins were irradiated (lambda > 300 nm, 12 mW/cm2) in the presence and absence of Xan. Upon irradiation and in the presence of Xan, lens proteins polymerized in the order alpha > beta > gamma as assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Further analysis of the photolyzed alpha-crystallin by mass spectrometry indicated that histidine, tryptophan and methionine residues were oxidized at specific positions in a dose-dependent (irradiation time) manner. In alpha A-crystallin two forms of oxidized histidine 154 were observed, 2-imidazolone and 2-oxohistidine. Our results suggest that naturally occurring Xan is a chromophore capable of photosensitization and photooxidation of lens proteins. Furthermore, this compound could play a role in age-related cataractogenesis.     

LINEAR DICHROISM STUDY OF METALLOPORPHYRIN TRANSITION MOMENTS IN VIEW OF RADIATIONLESS INTERACTIONS WITH TRYPTOPHAN IN HEMOPROTEINS

We measured the linear dichroism of several metalloporphyrins embedded in stretched polyvinyl alcohol (PVA) films to estimate the orientation of the absorption transition moments, which in hemoproteins are relevant to the radiationless energy transfer between tryptophan and heme. The metalloporphyrins were derivatives of protoporphyrin IX (PPIX), namely Fe³⁺-PPIX (ferric-heme) and Fe²⁺CO-PPIX (CO-heme), Mg-PPIX (Mg-heme) and Zn-PPIX (Zn-heme). Measurements were conducted between 300 and 700 nm. In all cases the linear dichroism was wavelength dependent, indicating the presence of several transition moments with different orientations. We focused our attention on the near-UV (300–380 nm) and Soret (380450 nm) absorption bands. Deconvolution in terms of Gaussian components gave three components between 380 and 450 nm and only one in the 300–380 nm region. Deconvolution of the near-UV and Soret spectra of oxy-, deoxy- and carbonmonoxyhemoglobin gave very similar results, suggesting a very similar orientation of the various transition moments in the free and protein-embedded hemes. It should be stressed that the single 300–380 nm band is the only one responsible for the overlap integral that regulates the energy transfer from tryptophan to heme in hemoproteins (Gryczynski et al., Biophys. J . 63, 648–653, 1992). The dichroism of this single band indicated that its transition moment is oriented at about 60 from the α-γ meso-axis of the heme moiety. We conclude that the heme should be considered a linear oscillator when it acts as acceptor of energy transfer from tryptophans.