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.     

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.     

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.     

Chaperone Function of Rat Lens α-Crystallin

A solution of γ-crystallin became turbid upon beating at 65 °C for 30 minutes; however, addition of α-crystallin suppressed this thermal aggregation. It was found the effective chaperone function could be achieved with the molar ratio of α/γ greater than 1/20. In terms of crystallin subunit, five molecular α-crystallin subunits could afford chaperone for one molecular γ-crystallin. The gel filtration profile of the sample solution, containing α- and γ-crystallins and preincubation at 65 °C for 30 minutes, showed complex formation between α- and γ-crystallins, indicating α-crystallin was bound to thermally denatured γ-crystallin. A 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence study showed that α-crystallin has more hydrophobic regions exposed after thermal incubation. In the presence of urea, both the α-crystallin chaperone activity and the ANS fluorescence intensity decreased. Accordingly, hydrophobic regions of α-crystallin play an indispensible role in its chaperone activity.     

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.     

Identifying the sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA) as a potential target for hypericin - a theoretical study

The exact cellular target for the potent anti-cancer agent hypericin has not yet been determined; this thus encourages the application of computational chemistry tools to be employed in order to provide insights that can be employed in further drug development studies. In the present study computational docking and molecular dynamics simulations are applied to investigate possible interactions between hypericin and the Ca(2+) pump SERCA as proposed in the literature. Hypericin was found to bind strongly both in pockets within the transmembrane region and in the cytosolic region of the protein, although the two studied isoforms of SERCA differ slightly in their preferred binding sites. The calculated binding energies for hypericin in the four investigated sites were of the same magnitude as for thapsigargin (TG), the most potent SERCA inhibitor, or in the range between TG and di-tert-butylhydroquinone (BHQ), which is also known to possess inhibitory activity. The hydrophobic character of hypericin indicates that the molecule initially binds in the ER membrane from which it diffuses into the transmembrane region of the protein and to binding pockets therein. The transmembrane TG and BHQ binding pockets provide suitable locations for hypericin as they allow for favourable interactions with the lipid tails that surround these. High binding energies were noted for hypericin in these pockets and are expected to constitute highly possible binding sites due to their accessibility from the ER membrane. Hypericin most likely binds to both isoforms of SERCA and acts as an inhibitor or, under light irradiation, as a singlet oxygen generator that in turn degrades the protein or induces lipid peroxidation.     

MONITORING LIGHT-INDUCED CHANGES IN ISOLATED, INTACT EYE LENSES

Fluorescence spectral changes occurring upon irradiation with 300 nm light have been monitored in situ in isolated, intact, whole lenses from the eyes of several species. The findings corroborate observations on other individual constituent protein molecules in the solution state, and also reveal features attributable to the supramolecular protein assembly that exists in the whole lens. Irradiation of the lens with 300 nm light causes red shifts in the tryptophan emission spectrum, suggesting alterations in the protein packing in the lens. Intermolecular energy transfer from tryptophan to one of the photoproducts, presumably N-formylkynurenine (N-FK), occurs in the condensed-phase sample. The N-FK formed is photodegraded efficiently in the lens, indicating that the photodynamic effects of endogenous N-FK might not be as severe as has been thought. Species variation in the photoevents are evident, particularly in avian lenses that contain the variant δ-crystallin as the core protein. The photoinduced changes in the near-UV circular dichroism of δ-crystallin (which is α-helical, as opposed to the β-sheet structure of α-, β-, and -γ-crystallins), isolated from chicken lenses, are remarkably different from other crystallins. Irradiation of δ-crystallin leads to a drastic reduction of circular dichroism intensity in the 250–300 nm region, whereas no changes are seen in the peptide absorption band.     

Revisiting the Rate-Limiting Step of the ANS–Protein Binding at the Protein Surface and Inside the Hydrophobic Cavity

8-Anilino-1-naphthalenesulfonic acid (ANS) is used as a hydrophobic fluorescence probe due to its high intensity in hydrophobic environments, and also as a microenvironment probe because of its unique ability to exhibit peak shift and intensity change depending on the surrounding solvent environment. The difference in fluorescence can not only be caused by the microenvironment but can also be affected by the binding affinity, which is represented by the binding constant (K). However, the overall binding process considering the binding constant is not fully understood, which requires the ANS fluorescence binding mechanism to be examined. In this study, to reveal the rate-limiting step of the ANS–protein binding process, protein concentration-dependent measurements of the ANS fluorescence of lysozyme and bovine serum albumin were performed, and the binding constants were analyzed. The results suggest that the main factor of the binding process is the microenvironment at the binding site, which restricts the attached ANS molecule, rather than the attractive diffusion-limited association. The molecular mechanism of ANS–protein binding will help us to interpret the molecular motions of ANS molecules at the binding site in detail, especially with respect to an equilibrium perspective.     

THE MOLECULAR CHAPERONE FUNCTION OF α-CRYSTALLIN IS IMPAIRED BY UV PHOTOLYSIS

Buffer solutions of the lens protein γ-crystallin and the enzymes aldolase and liver alcohol dehydrogenase became turbid and formed solid precipitate upon exposure to an elevated temperature of 63°C or to UV radiation at 308 nm. When α-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 α-crystallin. Hence, normal α-crystallin functioned as a molecular chaperone, providing protection against both UV and heat-induced protein aggregation. When α-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 α-crystallin was to cause interpeptide crosslinking among the αA₂ and αB₂ subunits of the α-crystallin macromolecule. In our experiments α-crystallin was exposed to UV doses, which resulted in 0, 50 and 90% crosslinking as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. α-Crystallin samples that were 50% and 90% crosslinked gave chaperone protection, which was increasingly impaired relative to unirradiated α-crystallin. The results are consistent with the notion that UV irradiation of α-crystallin results in loss of chaperone binding sites.     

Monitoring the Tanford transition in β‐lactoglobulin by 8‐anilino‐1‐naphthalene sulfonate and mass spectrometry

The fluorescent dye 8‐anilino‐1‐naphthalene sulfonate (ANS) is known to interact with proteins by conformation‐specific hydrophobic interactions and rather nonspecific electrostatic interactions. To which category the complexes detectable by mass spectrometry (MS) belong is still the subject of debate. Here, the Tanford transition in β‐lactoglobulin (BLG) is exploited as an experimental device to expose hydrophobic binding sites by an increase in pH, rather than, as usually done, by lowering the pH. Complex formation is monitored by electrospray ionization (ESI)‐MS and fluorescence spectroscopy. Both techniques reveal stronger ANS binding to BLG at pH 7.9 than at pH 5.9, suggesting that dye binding inside the calyx, which is known to be hydrophobically driven in solution, can contribute to the complexes detected by ESI‐MS. Electrostatic interactions between the protein and the ANS sulfonate group can only be weaker at pH 7.9 than at pH 5.9, supporting the interpretation of the results by the protein conformational change. Lysozyme is used as a negative control, which shows no variation in the interaction with ANS in the same range of pH, in the absence of conformational changes. However, comparison of MS and fluorescence data at variable pH for BLG and myoglobin (Mb) suggests that conformation‐specific ANS binding to proteins is detectable by ESI‐MS only inside well‐structured cavities of folded structures, like the BLG calyx and apoMb heme pocket. Indeed, ANS interactions with highly dynamic structures or molten globules, although detectable in solution, are easily lost in the gas phase. Copyright © 2008 John Wiley & Sons, Ltd.     

Binding of Organic Dyes with Human Serum Albumin: A Single‐Molecule Study

Kinetics of binding of dyes at different sites of human serum albumin (HSA) has been studied by single‐molecule spectroscopy. The protein was immobilized on a glass surface. To probe different binding sites (hydrophobic and hydrophilic) two dyes, coumarin 153 ( C153 , neutral) and rhodamine 6G ( R6G , cationic) were chosen. For both the dyes, a major (ca. 96‐98 %) and minor (ca. 3 %) binding site were detected. Rate constants of association and dissociation were simultaneously determined from directly measuring fluctuations in fluorescence intensity (τ_off and τ_on) and from this the equilibrium (binding) constants were calculated. Fluorescence lifetimes at individual sites were obtained from burst‐integrated lifetime analysis. Distributions of lifetime histograms for both the probes ( C153 and R6G ) exhibit two maxima, which indicates the presence of two binding domains in the protein. Unfolding of the protein has been studied by adding guanidinium hydrochloride (GdnHCl) to the solution. It is observed that addition of GdnHCl affects the dissociation and association kinetics and hence, binding equilibrium of the association of C153 . However, the effect of binding of R6G is not affected much. It is proposed that GdnHCl affects the hydrophobic binding sites more than the hydrophilic site.     

In Vitro Renaturation of Alkaline Family G/11 Xylanase via a Folding Intermediate: α-Crystallin Facilitates Refolding in an ATP-Independent Manner

In this study, alkaliphilic family G/11 xylanase from alkali-tolerant filamentous fungi Penicillium citrinum MTCC 6489 was used as a model system to gain insight into the molecular aspects of unfolding/refolding of alkaliphilic glycosyl hydrolase protein family. The intrinsic protein fluorescence suggested a putative intermediate state of protein in presence of 2 M guanidium hydrochloride (GdmCl) with an emission maximum of 353 nm. Here we studied the refolding of GdmCl-denatured alkaline xylanase in the presence and the absence of a multimeric chaperone protein α-crystallin to elucidate the molecular mechanism of intramolecular interactions of the alkaliphilic xylanase protein that dictates its extremophilic character. Our results, based on intrinsic tryptophan fluorescence and hydrophobic fluorophore 8-anilino-1- naphthalene sulfonate-binding studies, suggest that α-crystallin formed a complex with a putative molten globule-like intermediate in the refolding pathway of xylanase in an ATP-independent manner. A 2 M GdmCl is sufficient to denature alkaline xylanase completely. The hydrodynamic radius (R_H) of a native alkaline xylanase is 4.0, which becomes 5.0 in the presence of 2 M GdmCl whereas in presence of the higher concentration of GdmCl R_H value was shifted to 100, indicating the aggregation of denatured xylanase. The α-crystallin·xylanase complex exhibited the recovery of functional activity with the extent of ~43%. Addition of ATP to the complex did not show any significant effect on activity recovery of the denatured protein.     

Photosensitized Structural Modifications of the Lens Protein α‐Crystallin: Do All Modifications Impair Chaperone‐like Activity?¶

Among chaperone-like functioning proteins, the lens alpha-crystallins are of particular interest because they are not renewed, and even minor alterations can hurt their function of maintaining the proper refractive index and avoiding cataract formation in the lens. Several reports have suggested the occurrence of remarkable structural modifications in lens proteins in the presence of endogenous and exogenous sensitizers upon exposure to light. In particular, it has been shown in vitro that hypericin, the active ingredient of Hypericum, can bind to and, in the presence of light, cause the photopolymerization of alpha-crystallin. On the basis of these results it has also been suggested that a subsequent significant impairment of the protein function can occur. Using absorption and emission spectroscopic techniques, as well as circular dichroism, we have studied the structural modifications of alpha-crystallin resulting from its interaction with hypericin after irradiation with visible light. To investigate the chaperone-like function of alpha-crystallin, the heat-induced aggregation kinetics of another lens protein, betaLow-crystallin, was monitored by measuring the apparent absorption due to scattering at 360 nm as a function of time, and no apparent damage to its functional role was observed. Spectroscopic results, on the contrary, show a prominent reduction in both tryptophan and hypericin fluorescence emission intensity after light irradiation, suggesting an alteration in the tryptophan microenvironment and a high degree of packing of the chromophore due to photoinduced modification of the molecular framework. Control experiments on alpha-crystallin structurally modified by light in the presence of hypericin indicated that the protein still retains its ability to chaperone both lens crystallins and insulin.     

Nonspecific protein-DNA interactions: complexation of alpha-chymotrypsin with a genomic DNA

In this contribution, we report studies on nonspecific protein-DNA interactions of an enzyme protein bovine pancreatic alpha-chymotrypsin (CHT) with genomic DNA (from salmon testes) using two biologically common fluorescent probes: 1-anilinonaphthalene-8-sulfonate (ANS) and 2,6-p-toluidinonaphthalene sulfonate (TNS). TNS molecules that are nonspecifically bound to positively charged basic residues at the surface sites, not in the hydrophobic cavities of the protein, are preferentially displaced upon complexation of TNS-labeled CHT with DNA. The time-resolved fluorescence anisotropy of TNS molecules bound to hydrophobic cavities/clefts of CHT reveals that global tumbling motion of the protein is almost frozen in the protein-DNA complex. A control study on TNS-labeled human serum albumin (HSA) upon interaction with DNA clearly indicates that the ligands in the deep pockets of the protein cannot be displaced by interaction with DNA. We have also found that ANS, which binds to a specific surface site of CHT, is not displaced by DNA. The intactness of the ANS binding in CHT upon complexation with DNA offers the opportunity to measure the distance between the ANS binding site and the contact point of the ethidium bromide (EB)-labeled DNA using the F?rster resonance energy transfer (FRET) technique. Enzymatic activity studies on CHT on a substrate (Ala-Ala-Phe 7-amido-4-methyl coumarin) reveal that the active site of the enzyme remains open for the substrate even in the protein-DNA complex. Circular dichroism (CD) studies on CHT upon complexation with DNA confirm the structural integrity of CHT in the complex. Our studies have attempted to explore an application of nonspecific protein-DNA interactions in the characterization of ligand binding of a protein in solution.     

Mechanism of interaction of vincristine sulphate and rifampicin with bovine serum albumin: A spectroscopic study

The mechanism of interaction of vincristine sulphate (VS) and rifampicin (RF) with bovine serum albumin (BSA) has been studied by quenching of BSA fluorescence by RF/VS. The Stern-Volmer plot indicates the presence of a static component in the quenching mechanism. Results also show that both the tryptophan residues of BSA are accessible to VS and RF. The high magnitude of rate constant of quenching indicates that the process of energy transfer occurs by intermolecular interaction and VS/RF-binding site is in close proximity to the tryptophan residues of BSA. Binding studies in the presence of a hydrophobic probe, 8-anilino-1-naphthalene-sulphonic acid sodium salt (ANS) indicate that the VS and RF compete with ANS for hydrophobic sites on the surface of BSA. Small decreases in critical micellar concentrations (CMC) of anionic surfactants in presence of VS/ RF show that the ionic character of VS/RF also contributes to binding. The temperature dependence of the association constant is used to estimate the values of the thermodynamic parameters involved in the interaction of VS/RF with BSA and the results indicate that hydrophobic forces play a significant role in the binding. Circular dichroism studies reveal that the change in helicity of BSA are due to binding of VS/RF to BSA.     

Fluorescence studies on the interaction of hydrophobic ligands with Momordica charantia (bitter gourd) seed lectin

The interaction of Momordica charantia (bitter gourd) seed lectin (MCL) with several nucleic acid bases has been investigated by monitoring changes induced in the protein fluorescence by ligand binding. Values of the binding constant, K_a were obtained as 1.1 × 10⁴, 1.56 × 10⁴ and 2.2 × 10³ M^?1 for adenine, cytosine and uracil, respectively. In addition, binding of 8-anilinonaphthalene 1-sulfonate (ANS) with MCL was investigated by fluorescence spectroscopy. Interaction with MCL at low pH results in a large enhancement of the fluorescence intensity of ANS with a concomitant blue shift in the emission λ_max, whereas at neutral and basic pH changes in both fluorescence intensity and emission maximum were very small, clearly suggesting that the MCL–ANS interaction is stronger at lower pH values. When excited at 295 nm in the presence of ANS, the protein fluorescence decreased with a concomitant increase in the emission intensity of ANS, suggesting resonance energy transfer from the tryptophan residues of MCL to ANS. Gel filtration profiles of MCL at pH values 2.0 and 7.4 are similar indicating that the tetrameric nature of MCL is retained even at low pH. Addition of lactose or adenine to MCL–ANS mixture did not alter the change in ANS fluorescence suggesting that lactose, adenine and ANS bind to MCL at independent and non-interacting sites. These results are relevant to understanding the functional role of MCL in the parent tissue.     

Spectroscopic studies on the mode of interaction of an anticancer drug with bovine serum albumin

The mechanism of interaction of vinblastin sulphate (VBS) with bovine serum albumin (BSA) has been reported. Association constant for VBS-BSA binding was found to be 3.146+/-0.06 x 10(4) M(-1). Stern-Volmer analysis of fluorescence quenching data showed that the fraction of fluorophore (protein) accessible to the quencher (drug) was close to unity indicating thereby that both tryptophan residues of BSA are involved in drug-protein interaction. The rate constant for quenching, greater than 10(10) M(-1) S(-1), indicated that the drug-binding site is in close proximity to tryptophan residues of BSA. Binding studies in the presence of an hydrophobic probe, 8-anilino-1-naphthalein-sulphonic acid, sodium salt (ANS) indicated that there is hydrophobic interaction between VBS and probe and they do not share common sites in BSA. Thermodynamic parameters obtained from data at different temperatures showed that the binding of VBS to BSA involves predominant hydrophobic forces. The effects of some additives and paracetamol on binding of VBS-BSA have also been investigated. The CD spectrum of BSA in presence of VBS shows that the binding of VBS leads to change in the helicity of BSA.     

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.     

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.