Eiji Fujimori

CAROTENOID CHROMOPHORE LENGTH AND PROTECTION AGAINST PHOTOSENSITIZATION

Abstract— Carotenoid pigments were extracted and purified from wild‐type and mutants 7 and 93a of Sarcina lutea, and tested for their ability to quench 1O2. The wild‐type pigment (P‐438, 9 conjugated double bonds) is as active in quenching 1O2 as is β‐carotene. On the other hand, the pigment P‐422 (8 conjugated double bonds) from mutant 7 is 2 or 3 times less efficient, whereas phytofluene and phytoene from S. lutea are 100 and 1000 times less efficient, respectively, than is β‐carotene at quenching 1O2. It was also found that the broad EPR signal, induced by light in benzene solutions of chlorophyll a and hydroquinone, and related to chlorophyll oxidation, is efficiently quenched by P‐438 and to a much smaller extent also by Sarcina phytoene.

Cross-linking and fluorescence changes of collagen by glycation and oxidation

The non-enzymatic glucosylation of collagen in vivo and in vitro produces blue-fluorescent cross-links very slowly. The mechanism of their formation is unknown. We investigated the role of oxidation in glycation. When native fluorescent collagen from old-rat tail tendon and its CNBr peptides were oxidized by chemically generated singlet oxygen, cross-linking occurred immediately, and the cross-linked products showed an increased blue fluorescence. Further cross-linking and development of blue fluorescence also were accelerated by singlet oxygen when oxidizing in vitro glucosylated collagen CNBr peptides. It was noted that the blue fluorescence developed at the expense of a near-UV fluorescence. This near-UV fluorophore, which is also present in native collagen, was found to be produced by the in vitro glucosylation of collagen and during the cross-linking by glucosylation was slowly converted to the blue fluorophore. These changes indicate the autoxidation of near-UV fluorescent intermediates to blue fluorescent cross-links during glucosylation. Non-enzymatic fructosylation, which occurs in vivo in certain proteins, was more effective than glucosylation in forming fluorophores and cross-links with collagen in vitro. Fructosylated fluorophores were found different from glucosylated products in their oxidation reactivities with singlet oxygen.

Crosslinking and blue-fluorescence of photo-oxidized calf-lens α-crystallin

Abstract In the u.v. (300 nm)-induced photo-oxidation of calf-lens α-crystallin, the polypeptides are crosslinked and blue fluorescent products are formed. The formation of 440 460 nm blue-fluorescent species and the crosslinking of polypeptides are both inhibited by the presence of glutathione. When u.v. (300 nm)-irradiated α-crystallin is further irradiated with u.v. (365 nm) light, both the 440 460 nm fluorescent products and the crosslinking are increased. [The primary photoproduct N-formylkynurenine, which is produced by the first u.v. (300 nm) irradiation, can absorb u.v. (365 nm) light and is converted to its secondary photoproduct.] Thus the secondary photoproduct of N-formylkynurenine could be involved in both the 440 460 nm fluorescent chromophores and the crosslinking. Thermal stability studies suggest that these two 440 460 nm fluorescent chromophores may be different forms of the same secondary photoproduct.

Ultraviolet light- and ozone-induced changes in pyridinoline, a trisubstituted 3-hydroxypyridinium crosslink of collagen

Pyridinoline photo-degraded with the formation of photoproducts absorbing diffusely around 260-290 nm (pH 7) and sharply at 232 nm (pH 1). Subsequent heating partially regenerated the original pyridinoline, also producing new products absorbing at 417/440 nm (pH 7) and 300/412 nm (pH 1). Pyridinoline (pH 7) and its new products (pH 7 and pH 1) also underwent ozone-induced degradation.

Cross-linking of collagen CNBr peptides by ozone or UV light

Insoluble collagen from rat tail tendon was digested with cyanogen bromide. The resultant peptides were dissolved in 0.1% SDS solution and separated by gel filtration and gel electrophoresis. Cross‐linking occurred in CNBr‐cleaved peptides when they were exposed to ozone or biologically effective UV (300 nm) radiation. The enhancement of a blue fluorescence at 430 nm (excited at 350 nm) was found to be associated with oxidized, cross‐linked peptides. Polymeric peptides, formed in collagen with aging, also exhibited enhanced blue fluorescence.

Blue fluorescence and crosslinking of photooxidized proteins

Upon aging, ocular lens proteins increase crosslinking and visible fluorescence [ 11. The direct or sensitized photooxidation of tryptophan residues in proteins produces the primary photoproduct N-formylkynurenine (FK) [2,3], whose blue fluorescence_ excited at 350 nm emits at 400 and 435 nm [2,4]. In the UV (300 nm)-induced photooxidation of calflens cr-crystallin, we have discovered the formation of new blue fluorescence bands at 440 and 460 nm, which can be excited at 400-420 nm [4-61. When UV (300 nm)-irradiated acrystallin is further irradiated with UV (36.5 nm) light which is absorbed by FK, the 400/435 nm fluorescence is converted to the 440/460 nm fluorescence [S]. Furthermore, photooxidized a-crystallin is crosslinked, while subsequent W (365 nm)-irradiation increases crosslinking [6]. To compare with calf-lens cY-crystallin, other tryptophan-containing proteins have been investigated. A parallelism between crosslinking and 440/460 nm fluorescence has been found in other photooxidized proteins. In addition, 440/460 nm fluorescence and crosslinking were also found to be affected by hydroxylamine or hydrazine. gular quartz cell (1 cm X 1 cm) was irradiated for 2 h with W (300 nm) light from a Xe lamp (150 W), connected to a Bausch and Lomb high intensity monochromator (band width *lo nm), at 10 cm from the exit slit. For subsequent W (365 nm) irradiation for 1 h, a Hg lamp with a filter for the 365 nm Hg line was used at 20 cm from the filter (intensity 2.8-3 mW/cm2). Before and after W-irradiation, fluorescence (excited at 350 and 400 nm) and excitation (for 450 nm fluorescence) spectra were measured using a Perkin-Elmer fluorescence spectrophotometer model 650-10 S. Gel-electrophoresis experiments (6% acrylamide gel for myosin and BSA, 13% gel for other proteins) were also performed as in [6].

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.

pH-dependent photoreactions of blue-fluorescent calf α-crystallin

Abstract Upon irradiation of weakly blue-fluorescent calf α-crystallin with u.v.(300 nm)-light, changes in the fluorescence occur. An intense blue fluorescence at 400 nm (excitation at 330–350 nm) is predominantly produced at pH 10–11, whereas at pH 7·5–8·5 the formation of a less intense fluorescence at 435 nm (excitation at 350 nm) is more favorable than that of the 400 nm fluorescence. When blue-fluorescent α-crystallin at pH 10·5 is irradiated with u.v.(365 nm)-light, the 435 nm-fluorescence is converted to the 400 nm-fluorescence, as observed in u.v.(300 nm)-irradiated α-crystallin at pH 8·2. This finding supports the suggestion that the blue-fluorescent chromophores of α-crystallin are the same as photochemically-produced chromophores ( N -formylkynurenine). The above photo-conversion may be attributed to a photo-proton transfer (deprotonation) from the formamide to the ortho-carbonyl in N-formylkynurenine and is inhibited by glutathione. The u.v.(300 or 365 nm)-induced formation of other blue fluorescences at 440 and 460 nm (excitation at 400 nm) is also pH-dependent and inhibited by glutathione. When u.v.(300 nm)-irradiated α-crystallin is further irradiated with u.v.(365 nm)-light in the presence of ascorbic acid in alkaline solution, longer-wavelength fluorescences in the range of 460–510 nm (excitation at 420 nm) are produced.

CHLOROPHYLL‐PHOTOSENSITIZED OXIDATION OF HYDROQUINONE AND p‐PHENYLENEDIAMINE DERIVATIVES

Abstract— ESR and photovoltaic studies on light‐induced one‐electron transfer between chlorophyll a and electron donors in the absence of oxygen show (1) the possible conversion of photo‐reduced chlorophyll a and p‐benzosemiquinone ion radicals to their non‐ionic radicals in methanol solutions of low pH, (2) the production of ESR absorption of tetrachloro‐p‐benzosemi‐quinone even in benzene, enhanced by the addition of triethylamine or methanol, and (3) the transfer of one electron from tetramethyl‐p‐phenylenediamine to either excited chlorophyll a or pheophytin a in methanol at pH above 3.6 but not to pheophytin a at pH below 1 0 where its radical cation appears to accept an electron from excited pheophytin a. Bacteteriochloro‐phyll is also shown to be capable of photooxidizing hydroquinones and tetramethyl‐p‐phenyl‐enediamine.

Blue-fluorescent bovine α-crystallin

Abstract Blue-fluorescent α-crystallin has been isolated from bovine lenses by gel-filtration on Sephacryl S-200 Superfine. The blue fluorescence of this α-crystallin is characterized by fluorescence peaks at about 410 and 435 nm and two excitation peaks at about 350 and 370 nm. This finding suggests the existence of two different blue-fluorescences in bovine α-crystallin. Both low molecular weight α-crystallin and higher molecular weight α-crystallin exhibit similar blue fluorescence. With aging, in the nuclear region of bovine lenses, blue-fluorescent low molecular weight α-crystallin shifts to non-covalently-linked higher molecular weight aggregates which are also blue-fluorescent.