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.     

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.     

Effect of the UV Modification of α-Crystallin on Its Ability to Suppress Nonspecific Aggregation

Recent studies have shown that structural modifications of α-crystallin during lens aging decrease it's effectiveness as a molecular chaperone. Some of these posttranslational modifications have been linked to UV radiation, and this study was undertaken to investigate the effect of UV irradiation on the ability of α-crystallin to suppress nonspecific aggregation. The effect of 3-hydroxykynurenine (3-HK) was also investigated as a model for its glucoside (3-HKG), a main lens chromophore that has been linked to photochemical changes in the human lens. Alpha- and γ-crystallin solutions (1 mg/mL, 1:0.125 wt/wt) were photolyzed (transmission above 295 nm) for various time intervals. Thermal denaturation of γ-crystallin with or without α-crystallin was carried out at 70°C and increases in light scattering were measured at 360 nm. We found that (1) irradiation of γ-crystallin increased its susceptibility to heat-induced scattering. The addition of α-crystallin protects it against thermal denaturation, although its ability to do so decreases the longer γ-crystallin is irradiated and (2) irradiation of α-crystallin decreases its ability to suppress nonspecific aggregation and the presence of 3-HK during irradiation decreases it further. Our results indicate that posttranslational modifications of α-crystallin due to UV irradiation affect the sites and mechanisms by which it interacts with γ-crystallin. The kinetics of γ-crystallin unfolding during thermal denaturation were also analyzed. We found that a simple two state model applies for nonirradiated γ-crystallin. This model does not hold when γ-crystallin is irradiated in the presence or absence of α-crystallin. In these cases, two step or multistep mechanisms are more likely.     

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.     

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.     

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.     

Photochemistry and Thermal Decarboxylation of α-Phosphoryloxy-p-nitrophenylacetates

α-Carboxy-4-nitrobenzyl phosphate 4 and its derived monomethyl phosphate ester 5 were synthesized and purified by anion-exchange chromatography. A gradient of LiCl was necessary for elution of the anion-exchange column to avoid unexpected thermal decarboxylation that occurred during vacuum evaporation when the volatile triethylammonium bicarbonate buffer was used. Photolysis of each compound was accompanied by decarboxylation, and 4 released inorganic phosphate with near-100% stoichiometry. Time-resolved infrared spectroscopy of the photolysis reaction, coupled with density functional theory calculations of vibrational frequencies, enabled us to infer a mechanism for the photolytic pathway, although there was some evidence for a second pathway also being operative. In contrast to the results for 4 , photolysis of 5 appeared to release little or no monomethyl phosphate.     

GLOBAL ANALYSIS OF THE TIME-RESOL VED FLUORESCENCE OF α-CHYMOTRYPSINOGEN A AND α-CHYMOTRYPSIN POWDERS AS A FUNCTION OF HYDRATION

The time-resolved tryptophyl fluorescence of alpha-chymotrypsin A and alpha-chymotrypsin in the crystalline state and in buffer solution at room temperature was analyzed globally. Triple-exponential decay functions are necessary to adequately describe the tryptophyl fluorescence decay surfaces of the protein powders as a function of hydration and in solution. The fluorescence lifetimes of alpha-chymotrypsinogen A (tau 1 = 0.32, tau 2 = 1.30 ns, tau 3 = 3.98 ns) and alpha-chymotrypsin(tau 1 = 0.66 n s, tau 2 = 2.26 ns, tau 3 = 5.40 ns) are constant over the entire hydration range. The spectral positions of the decay-associated spectra of the hydrated powders do not shift as a function of hydration. This indicates that the structures of the zymogen and the active enzyme are unaffected by hydration. The lifetimes of alpha-chymotrypsinogen A in phosphate buffer pH 7.4 are tau 1 = 0.37 ns, tau 2 = 1.17 ns and tau 3 = 3.44 ns while the respective values of alpha-chymotrypsin are tau 1 = 0.47 ns, tau 2 = 1.40 and tau 1 = 3.89 ns.     

α-TERTHIENYL PHOTOSENSITIZES DAMAGE TO pBR322 DNA

alpha-Terthienyl photosensitizes single strand breaks in pBR322 DNA. Almost identical results were observed under oxygen and under argon. In the presence of oxygen, this DNA nicking was enhanced by histidine and was not affected by superoxide dismutase, catalase, or the antioxidant BHT. Although chemical damage to DNA treated with alpha-terthienyl plus near-UV was clearly demonstrated in vitro, transformation in E. coli with this damaged pBR322 DNA still took place. Likewise, Haemophilus influenzae DNA transforming activity was not significantly decreased by photosensitization with alpha-terthienyl.     

LOW-TEMPERATURE TRAPPING OF EARLY PHOTOINTERMEDIATES OF α-ISORHODOPSIN

Abstract— a-Isorhodopsin, an artificial visual pigment with a 9- cis -4,5-dehydro-5,6-dihydro(a)retinal chromophore, was photolyzed at low temperatures and absorption difference spectra were collected as the sample was warmed. A bathorhodopsin (Batho)-like intermediate absorbing at ca 495 nm was detected below 55 K, a blue-shifted intermediate (BSI)-like intermediate absorbing at ca 453 nm was observed when the temperature was raised to 60 K and a lumirhodopsin (Lumi)-like intermediate absorbing at ca 470 nm was found when the sample was warmed to 115 K. Photointermediates from this pigment were compared to those of native rhodopsin and 5,6-dihydroisorhodopsin. As in native rho-dopsin, Batho is the first intermediate detected in a-isorhodopsin, though unlike native rhodopsin at low temperatures BSI is observed prior to Lumi formation. a-Isorhodopsin behaves similarly to 5,6-dihydroisorhodopsin, with the same early intermediates observed in both artificial visual pigments lacking the C₅-C₆ double bond. The transition temperature for BSI formation is higher in a-isorhodopsin, suggesting an interaction involving the chromophore ring in BSI formation. The transition temperature for Lumi formation is similar for these two pigments as well as for native rhodopsin, suggesting comparable changes in the protein environment in that transition.     

ANTIVIRAL ACTIVITY OF THE PHOTOACTIVE THIOPHENE α-TERTHIENYL

The naturally occurring thiophene, α-terthienyl, was investigated for phototoxicity against several viruses and a line of mouse cells. The compound was extremely phototoxic to the two-membrane-containing animal viruses, murine cytomegalovirus (MCMV) and Sindbis virus (SV). Antiviral activity was detected at 10⁵μg/m in the presence of UVA. However, no effect was seen in the absence of UV-A, even at 0.1 μg/m of αT. Mouse cells were much more resistant to αT, as was the bacterial virus T4, which does not contain a membrane. Murine CMV, which had been inactivated by αT and UVA, penetrated mouse cells efficiently; but the viral DNA could not replicate, and late viral proteins were not made. Thus viral gene expression was inhibited in the photoinactivated virus. In order to account for all these data we suggest that αT may interact with viral proteins in addition to membrane lipids.     

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.     

Synthesis and Antiviral Activity of Modified 5α-Steroids

Chemistry of Natural Compounds -     

α-氯代脂肪酸对某些稀土的萃取研究

Гавриловаа等^[1]曾报导用α-溴代己酸的氯仿溶液对钐、钕、钆和镥的萃取,其萃取曲线与无取代基的比较向酸性区移动了约1个pH单位。Preston报导了α-溴癸酸的二甲苯溶液对一系列金属离子的萃取,认为所试验的8个稀土金属与α-溴癸酸形成了不溶性的金属络合物^[2]。     

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.     

PHOSPHORESCENCE SPECTRA OF α-DIKETONES IN LOW TEMPERATURE GLASSES

Abstract— The emission properties of o -anisil, p -anisil, benzil, and biacetyl in glasses containing varying proportions of isopropanol have been studied. The different kinds of phosphorescence spectra observed are proposed to be due to triplet states of different conformations and to triplet states of solvated species or other compounds formed by a reaction of the α-diketones with isopropanol.     

THERMAL GENERATION OF ELECTRONICALLY EXCITED STATES BY α-PEROXYLACTONES: ACTIVATION PARAMETERS AND EXCITATION YIELDS AS A FUNCTION OF α-DEUTERATION*

Abstract— The kinetics and the direct, the 9,10-dibromo- (DBA) and the 9,10-diphenylanthracene- (DPA) enhanced chemiluminescence of tert-butyl α-peroxylactone 1, with and without α-deuteration, was investigated in order to probe the mechanism of direct chemiexcitation by these hyperenergetic molecules. The small secondary isotope effect on the rates and activation parameters suggests that a diradical mechanism is obtained. The partitioning of the diradical intermediate into excited vs ground state product and the yield of triplet excited product are moderately (ca. 5-fold) increased by α-deuteration, but the singlet yield is unaffected. A convenient and useful chemiluminescence method for the determination of fluorescence quantum yields has been developed.