SENSITIZER-INDUCED CONFORMATIONAL CHANGES IN LENS CRYSTALLIN—I. PHOTODYNAMIC ACTION OF METHYLENE BLUE AND N-FORMYLKYNURENINE ON BOVINE α-CRYSTALLIN

Abstract— Fluorescence and circular dichroic properties of bovine a-crystallin have been monitored to detect changes in the structural integrity of the protein following photoreactions in the presence of sensitizer, either methylene blue or N-formylkynurenine. Methylene blue-sensitized photooxidation causes a change in the tertiary structure as manifested in the near-UV CD; this is observed within 0.5 h of irradiation during which time tryptophan emission decreases rapidly. Using inhibitors specific for active species of oxygen, it has been shown that singlet oxygen predominantly causes this change but the sensitizer molecules also have some role in this process. Upon 6 h of irradiation in the presence of methylene blue under both aerobic and anaerobic conditions, the thiol groups that were in a non-polar region of the protein are exposed to polar environments. In conformity with these fluorescence results. near-UV CD (tertiary structure) suffers a drastic alteration whereas the far-UV CD (secondary structure) remains virtually unchanged. The studies with inhibitors indicate that sensitizer molecule itself is primarily responsible for this process. This major change in the conformation has been explained by suggesting that a large portion of the protein unfolds in the photosensitized reaction, thereby altering microenviron-ments, orientation, and intermolecular interactions of different amino acids. N-formylkynurenine also shows some changes in the near-UV CD, presumably, caused by H₂O₂ generated in the photosensitized reaction. But the major alteration in the microenvironments of thiol groups and in the near-UV CD, as observed in the case of methylene blue, does not occur even when the protein is irradiated for 6 h in the presence of N-formylkynurenine and air.     

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.     

MOLECULAR CHANGES DURING THE PHOTOOXIDATION OF α-CRYSTALLIN IN THE PRESENCE OF UROPORPHYRIN

Singlet oxygen reacts preferentially with three amino acids in proteins, His, Trp and Met. In order to study the specific molecular events that result from such oxidations, calf a-crystallin was photooxidized in the presence of uroporphyrin and the reactions were investigated by high performance liquid chromatography peptide mapping using a photodiode array detector followed by fast atom bombardment mass spectrometry (FAB-MS). From these studies, the following conclusions can be inferred: (1) Upon photooxidation residue Met-68 of the B chain is oxidized to Met sulfoxide, whereas residue Trp-60 remains intact. (2) Two of the 16 His residues in a-crystallin are photooxidized with an apparent pK_a of ca 7.0. (3) FAB-MS analysis suggests that residue Lys-166 close to the C-terminal end of the A chain forms a cross-link with the His-7 residue close to the N-terminal end of the A chain. This may be either an inter- or intramolecular cross-link.     

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.     

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.     

THE ANAEROBIC PHOTOLYSIS OF LENS α-CRYSTALLIN: EVIDENCE FOR TRIPLET STATE MEDIATED PHOTODAMAGE

Anaerobic solutions of lens alpha-crystallin were subjected to near-UV (greater than 295 nm) irradiation, and the photoproducts were analyzed by fluorescence and room-temperature phosphorescence spectroscopy. The principal photoproduct was excited maximally at 340 nm, fluoresced maximally at 430 nm, and phosphoresced with an emission maximum at 510 nm. The phosphorescence intensity decay of this species was well fit by a sum of two exponentials with lifetimes of 9.2 ms (78%) and 61 ms (22%); this report is the first demonstration of a long-lived triplet state associated with a protein photolysis product. As reported previously, 3trp* is also long-lived in deoxygenated alpha-crystallin solution at room-temperature (Berger and Vanderkooi, 1989, Biochemistry 28, 5501-5508), hence both tryptophan and photoproduct triplet states are good candidates to mediate photodamage. Photolysis experiments in the presence of agents known to alter the tryptophan triplet yield provide evidence for the importance of triplet-state-mediated photodamage of lens crystallins in anaerobic solution. In 30 mM acrylamide where 3trp*, but not 1trp*, is efficiently quenched, anaerobic solutions exhibited marked resistance to protein photodamage, whereas the photoprotection in aerobic solution was minimal. In D2O, where photoionization is suppressed but triplet states are longer-lived, photodamage was accelerated in anaerobic solution but reduced in aerobic solutions. Finally, the anaerobic photodestruction rate was increased in 500 mM Cs+ solution where the triplet yield is increased by a heavy atom effect.     

MODE OF ACTION OF α-TERTHIENYL ON ESCHERICHIA COLI: EVIDENCE FOR A PHOTODYNAMIC EFFECT ON MEMBRANES

The photodynamic effects of α-terthienyl (αT) in near-UV light (UV-A) on Escherichia coli showed close agreement with the light absorption of αT at different wavelengths suggesting that αT is the primary absorbing molecule responsible for the photosensitized reaction. Studies with DNA repair deficient mutants of E. coli indicated that the bactericidal action of αT/UV-A was not mediated by DNA damage, in direct contrast to the well-known photosensitizer, 8-methoxypsoralen. By using a closed borosilicate glass reaction vessel and various gas mixtures, it was demonstrated that photosensitization of both E. coli and a more resistant bacterium, Pseudomonas aeruginosa , was absolutely dependent on the presence of oxygen. The rate of killing by αT/UV-A showed a rather small dependence on preincubation temperatures, with quite rapid killing at 5°C, suggesting that the movement of αT across the cytoplasmic membrane of E. coli is not the rate limiting step in killing and perhaps is not even necessary for killing. Sodium dodecyl sulphate-polyacrylamide gels of cell membrane proteins after 15 and 30min of treatment with αT/UV-A showed substantial random crosslinking of these proteins. The results taken overall suggest that αT is a photodynamic photosensitizer which exerts its primary effect at the level of the cytoplasmic membrane.     

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.     

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.     

PHOTODYNAMIC ACTION OF HEMATOPORPHYRIN ON YEAST CELLS—A KINETIC APPROACH

Abstract— By a technique which combines rapid mixing of cells and hematoporphyrin (HP) with a short duration of illumination, the photodynamic inactivation of yeast cells was investigated, particularly, in seeking for the information of the location of HP at the time of action. The fluence-survival curves obtained under the conditions where the reaction mixture was kept in the dark for Is, 60s and even 35 min before illumination were indistinguishable from each other, indicating no interaction between cells and sensitizers took place in about 30 min in such a way that the photodynamic efficiency could be modified. It is unlikely that HP acted intracellularly, since the protective effect of N^?₃ was observed at concentrations as low as 0.5 mM. Furthermore, the rate constant k_p related to the protective effect of NJ, was estimated to be 1 × 10⁸M^?1 s^?1 under the assumption that ¹O₂ was the active intermediate and had a lifetime of 2 μs under the present conditions. This value of k_p is rather close to that of k_q, the quenching rate constant of N^?₃ for ¹O₂, of which the accepted value is 2 × 10⁸M^?1s^?1 in the homogeneous aqueous system. This information, together with the absence of uptake of HP by cells and a well response of survival upon illumination to the D₂O fraction of the reaction mixture, provide strong bases for the argument that direct interaction of HP with yeast cells is of minor importance in the photodynamic processes, and the photodynamic action is largely mediated by an intermediate (¹0₂) generated in bulk medium.     

EFFECT OF SODIUM AZIDE ON PHOTODYNAMIC INDUCTION OF GENETIC CHANGES IN YEAST

Abstract— In view of the increasing attention to ¹O₂ (¹Δ_g) participation in the photodynamic action, different types of genetic changes in Saccharomyces cerevisiae by acridine orange sensitization were compared with respect to the response to N₃^-, a well known quencher of ¹O₂. The induction of mitotic crossing over with respect to ade 2 locus and mitotic gene conversion at trp 5 locus were suppressed by the addition of N₃^- suggesting the involvement of ¹O₂ as a major intermediate. However, the induction of reverse mutation at ilv 1 was only slightly suppressed. These results may indicate that there are two types of photodynamic DNA damage; one is produced via ¹O₂ and the other via non-¹O₂ reaction pathway which lead to mitotic gene conversion and mitotic crossing over, and to mutation, respectively.     

PHOTOREGULATION OF α-CHYMOTRYPSIN ACTIVITY BY ISOMERIZABLE INHIBITORS. STUDY OF THE ACTION MECHANISM*

Abstract— The N- p -phenylazophenyl-N-phenylcarbamyl chloride (PAPC) in its cis form is five times more active as inhibitor of α-chymotrypsin than in the trans form. In the present work, derivatives of PAPC have been synthesized. Each of these new compounds is photoisomerizable and is an inhibitor (in the cis and in the trans form) of α-chymotrypsin. The cis isomer is always more active than the trans. The m -methyl derivative is 17.5 times more active in the cis form than in the trans , whereas, for the para -substituted compound, this ratio is only 3.5.
Several hypotheses can explain this difference of activity between the cis and trans isomers: (1) steric hindrance towards the trans isomer, (2) lower affinity of the enzyme for the trans isomer, (3) higher reactivity of the complex formed between the enzyme and the cis form of the inhibitor. These hypotheses are discussed.     

DETECTION OF PORPHYRIN EXCITED STATES IN THE INTACT BOVINE LENS

Previous steady state and time resolved spectroscopic studies on porphyrins have shown that the triplet lifetimes of those sensitizers that bind to lens proteins are lengthened by several orders of magnitude. Presented here is an extension of this experiment to measure these transients in an intact bovine lens. As demonstrated by steady state fluorescence spectroscopy and flash photolysis, mesotetra (p-sulfonatophenyl)porphyrin (TPPS) binds to lens proteins. In air-saturated aqueous solution, TPPS has a triplet lifetime of 2 microseconds. In an intact bovine lens the triplet state decayed via biexponential kinetics with lifetimes of 0.16 and 1.6 microseconds. In addition to a lengthening of the lifetime there was a red shift in the triplet transient spectra of 10-20 nm of the porphyrin in the intact lenses.     

UPTAKE AND BIOCONVERSION OF α-TOCOPHERYL ACETATE TO α-TOCOPHEROL IN SKIN OF HAIRLESS MICE

Abstract— The photoprotective effect of topically applied α-tocopheryl acetate (vitamin E acetate), a stable derivative of α-tocopherol (vitamin E), and its possible bioconversion to the active antioxidant species (α-tocopherol) was examined in skin tissue of female hairless mice (HRS/J) exposed to UV-B irradiation. Our results indicate that topically applied α-tocopheryl acetate is absorbed into and retained by skin tissue. Furthermore, skin tissue from UV-B-irradiated animals that received daily topical α-tocopheryl acetate treatments contained significantly higher levels (P < 0.001) of α-tocopheryl acetate than non-UV-B-irradiated mice that received identical daily topical α-tocopheryl acetate treatments. Finally, free α-tocopherol levels in skin also were significantly increased (P < 0.00 1) by topical applications of α-tocopheryl acetate and skin levels of free α-tocopherol were significantly greater (P < 0.001) in UV-B-irradiated animals that received daily topical α-tocopheryl acetate treatments than in non-UV-Birradiated animals. These results suggest that UV-B irradiation enhances both the absorption of α-tocopheryl acetate and its bioconversion to free α-tocopherol.     

PHOTOFRAGMENTATION OF α-OXOCARBOXYLIC ACIDS IN AQUEOUS SOLUTION. AN EPR STUDY. FORMATION OF SEMIDIONE RADICALS BY DECARBOXYLATIVE SUBSTITUTION OF α-OXOCARBOXYLIC ACIDS BY ACYL RADICALS-I: GLYOXYLIC, PYRUVIC, α-OXOBUTYRIC, α-OXOGLUTARIC AND α-OXOISOCAPROlC ACIDS

Abstract— By means of in situ photolysis EPR of aqueous solutions of α-oxocarboxylic acids (RCO-CO₂H) at pH values above 5, semidione radical anions [RC(O^-)=C(O')R] and α-hydroxy-α-carboxy alkyl radicals [RC(OH)CO₂-] were detected. C0₂ was identified as a reaction product. On photolysis of mixtures of α-oxocarboxylic acids (RCOCO₂H and R'COCC₂H), "mixed" semidione radical anions [RC(O^->=C(O)R'] were observed in addition to RC(O^-)=C(O')R, R'C(O^-)=C(O')R', RC(OH)CO₂^- and R'C(OH)CO₂^-. The experimental results are explained in terms of photodecarboxylation (α-clea-vage) of electronically excited RCOCOJ to yield RCO and CO₂. The radicals RC(OH)CO₂^- are formed by reduction of RCOCO₂^- by CO₂^-. The semidione radicals are produced by addition of RCO to RCOCO₂^- followed by decarboxylation of the intermediate adduct. This mechanism was confirmed by generating acyl radicals independently and reacting them with α-oxocarboxylic acids. Selected product studies support the mechanism suggested.     

FORMATION OF REARRANGEMENT PRODUCT IN PHOTOLYSIS OF α,α-DIMETHYLDEOXYBENZOIN UPON CYCLODEXTRIN COMPLEXATION

Abstract Solid-state irradiation of cyclodextrin complexes of α,α-dimethyldeoxybenzoin results in the formation of a significant amount of rearrangement product, 4-isopropylbenzophenone, in addition to cage products. This behavior is not observed in the photolysis in solution or in micellar media.     

STUDIES ON FLAVINS IN ORGANIC SOLVENTS—II*. PHOTODECOMPOSITION OF RIBOFLAVIN IN THE PRESENCE OF OXYGEN

Abstract— The photodegradation of riboflavin by 436 mμ monochromatic light and of lumichrome by white nonfiltered light was studied in a set of organic solvents including ethanol, acetone, dioxane, pyridine and acetic acid. For comparison, water was used as a solvent. Photolysis was carried out in the presence of atmospheric oxygen. Riboflavin and lumichrome were found to be effectively stabilized towards the action of light by hydrogen bonds with solvent molecules, and consequently are most light-stable in water solutions. The overall scheme of riboflavin photolysis in organic solvents seems to be the same as in aqueous solutions. Lumichrome has been found as the main product of riboflavin photolysis in the organic solvents tested.     

ON THE LUMINESCENCE OF α-DIKETONES OF THE STEROID SERIES

Abstract—The absorption and luminescent properties of two steroids [16-ketoestrone (I), 5α-androstane-3β-ol-16,17-dione (II)] are compared. The behaviour of these compounds which differ only in their A-ring is significantly different with regard to solvent dependence. The transfer of excitation energy in compound (I) from the phenolic to the diketonic chromophore is studied and compared with data from analogous estrogens. The results obtained in this study indicate a significant long range (presumably conformational) influence of the steroid A and D rings on each other.     

ON THE QUENCHING OF SINGLET OXYGEN BY α-TOCOPHEROL

Abstract —D-α-tocopherol was found to be an effective quencher of ¹O₂ molecules ( k = 2.5 times 10⁸→mol^-1 s^-1 in pyridine) by measuring its effect on the autosensitized photooxidation of rubrene. The quenching process was shown to be almost entirely 'physical', that is, α-tocopherol deactivated about 120 ¹O₂ molecules before being destroyed. The results suggest that this process may be a mechanism for the protective effect of α - tocopherol in photodynamic action.