Light-driven transmembrane proton pumping by bacteriorhodopsin occurs in the photochemical cycle, which includes a number of spectroscopically identifiable intermediates. The development of methods to crystallize bacteriorhodopsin have allowed it to be studied with high-resolution X-ray diffraction, opening the possibility to advance substantially our knowledge of the structure and mechanism of this light-driven proton pump. A key step is to obtain the structures of the intermediate states formed during the photocycle of bacteriorhodopsin. One difficulty in these studies is how to trap selectively the intermediates at low temperatures and determine quantitatively their amounts in a photosteady state. In this paper we review the procedures for trapping the K, L, M and N intermediates of the bacteriorhodopsin photocycle and describe the difference absorption spectra accompanying the transformation of the all-trans-bacteriorhodopsin into each intermediate. This provides the means for quantitative analysis of the light-induced mixtures of different intermediates produced by illumination of the pigment at low temperatures.

Triflusal is a platelet antiaggregant drug with photoallergic side effects. However, it is considered a prodrug since it is metabolized to 2-hydroxy-4-trifluoromethylbenzoic acid (HTB)—the pharmacologically active form. HTB was found to be photolabile under various conditions. Its major photodegradation pathway appears to be the nucleophilic attack at the trifluoromethyl moiety. The involvement of the triplet state in the photodegradation has been unequivocally proved by direct detection of this transient in laser flash photolysis and by quenching experiments with oxygen, cyclohexadiene and naphthalene. Finally, the photobinding of HTB to proteins such as bovine serum albumin has been demonstrated using ultraviolet–visible (UV–Vis) and fluorescence spectroscopy. Nucleophilic groups present in the protein appear to be responsible for the formation of covalent drug photoadducts, which is the first step involved in the photoallergy shown by triflusal.

The effect of a π-electron–donating macrocyclic molecule on the photochromic behavior of viologen derivatives was investigated in a thin polymer film. The intermolecular interactions between the viologens and the macrocyclic molecule were investigated in a solution before photoirradiation. In acetone, benzylviologens, N,N′-dibenzyl-4,4′-bipyridinium hexafluorophosphate (1) and N,N′-dibenzyl-trans-1,2-bis(4-pyridinium)ethylene hexafluorophosphate (2) each derived from 4,4′-bipiyridine and trans-1,2-bis(4-pyridyl)ethylene, respectively, form an inclusion complex with p-benzocrown ether (3) with binding constants of ca 200 M⁻¹, which was driven by a charge transfer interaction. The peak wavelength of the charge transfer absorption band was at 453 and 421 nm for the inclusion complexes of 1 and 2 with 3, respectively. Upon photoirradiation to the polymer film containing 1, the film changed color from colorless to blue, associated with the reduction of 1 from the dication to the radical cation. The original dication was recovered after 120 min. The addition of 3 into the film containing 1 caused not only the color change from colorless to yellow, associated with the charge transfer interaction between 1 and 3 before photoirradiation, but also an acceleration in the bleaching rate of the photoreduced 1. When p-dimethoxybenzene (4) was used as an acyclic analog of 3, a negligible change in the photochromic behavior of 1 was observed. Similar effect of 3 on the photochromic behavior of 2 was observed. These results imply that the π-electron–donating macrocyclic molecule causes a faster bleaching of photoreduced viologens by forming the inclusion complex.

A perfluorinated derivative of phthalocyanine was synthesized as the free base, hexadeca-(2,2,2-trifluoroethoxy) phthalocyanine (H₂F₄₈Pc), and as a zinc complex, hexadeca-(2,2,2-trifluoroethoxy)-phthalocyaninatozinc (ZnF₄₈Pc), and their spectroscopic and photochemical properties were studied. The absorption bands are shifted bathochromically relative to simple phthalocyanines, exhibiting the longest wavelength band near 735 nm (H₂F₄₈Pc) and 705 (ZnF₄₈Pc). The solvatochromism of both compounds was modeled by Reichardt's E_T(30) parameter and Kamlet, Abboud and Taft multiparameter approach. The former, simpler, model was found to be adequate. We found that H₂F₄₈Pc undergoes unique basic and acidic titrations in organic solvents. These titration processes are accompanied by spectral changes that are explained on the basis of the chromophore's symmetry. Singular value decomposition was employed to resolve the spectra into the contributions of the species at various stages of protonation and to obtain the equilibrium constants. Nuclear magnetic resonance spectra (¹H, ¹⁹F and ¹³C) for the free base were obtained in a tetrahydrofuran-d₈ solution. The carbon spectrum, taken as a function of temperature, provided evidence for the presence of a tautomerization process, which switches the two internal hydrogens between the four central nitrogen atoms. As far as we know, this is the first report of the measurement of the free energy of activation for such process (ΔG^† = 10.6–11.4 kcal mol⁻¹ between 217 and 330 K) for a phthalocyanine, in solution. Like most other phthalocyanines these two compounds also act as photosensitizers and as generators of singlet molecular oxygen. The absolute quantum yields (Φ_Δ) for ZnF₄₈Pc was 0.58 ± 0.01 in benzene and 0.35 ± 0.01 in lipid vesicles. H₂F₄₈Pc had lower yields, 0.16 and 0.005, respectively. Either protonation or deprotonation of the pyrrole nitrogens in H₂F₄₈Pc lowered the Φ_Δ.

Hypocrellin B (HB), a lipid-soluble natural pigment of perylenequinone derivative, is considered as potential photosensitizer for photodynamic therapy. Liposomes loaded with HB can constitute a simple model system, appropriate for better understanding the photodynamic action of HB in vivo. The steady-state absorption and emission spectra, quantum yield and lifetime of fluorescence of HB incorporated into egg l-a-phosphatidyl-choline (EPC) liposome were examined. The photochemical properties (Type I and/or Type II) of HB have also been studied in aqueous dispersions of small unilamellar liposomes of EPC using electron paramagnetic resonance and spectrophotometric methods, respectively. The quantum yield of ¹O₂ generated by HB is ca 0.76 in chloroform solution and it did not change upon the incorporation of HB into liposomes of EPC. The superoxide anion radical was generated by the electron transfer from the anion radical of HB (HB^·−) to oxygen. The disproportionation of O₂^·− can generate H₂O₂ and ultimately the highly reactive ^·OH via the Fenton reaction. It could be that the disproportionation proceeded too fast, so we could not detect O₂^·− directly in aqueous dispersions of liposome EPC. Moreover, the self-sensitized photooxygenation of HB embedded in liposomes was studied, and almost fully (87%) inhibiting this reaction of HB by p-benzoquinone (as the quencher of O₂^·−) in aqueous dispersion of liposome EPC indicated that the radical mechanism (Type I) might be mainly involved in this oxygenation. All these findings suggested that the photodynamic action of HB proceeded via both Type-I and -II mechanisms, but Type-I mechanism might play a more important role in the aqueous dispersion.

The time-resolved absorption spectrum of singlet oxygen [O₂(a¹Δ_g) → O₂(b¹Σ_g )] has been recorded in the region ∼5100–5300 cm⁻¹ from air-saturated polystyrene samples using a microscope attached to a step-scan Fourier transform IR spectrometer. Singlet oxygen signals were observed with a time resolution of ∼160 ns from sample volumes of ∼20 nL using moderate data-acquisition times. These data indicate that it is reasonable and worthwhile to consider the further development of a transmission microscope as a viable tool to create singlet oxygen images of inhomogeneous samples including samples of biological importance.

Near-ultraviolet light (UVA: 320–400 nm) constitutes a major part of sunlight UV. It is important to know the effect of UVA on the biological activities of organisms on the earth. We have previously reported that black light induces somatic-cell mutation in Drosophila larvae. To investigate which wavelength of the UVA is responsible for the mutation we have now carried out a series of monochromatic irradiations (310, 320, 330, 340, 360, 380 and 400 nm) on Drosophila larvae, using the large spectrograph of the National Institute for Basic Biology (Okazaki National Research Institutes, Okazaki, Japan). Mutagenic activity was examined by the Drosophila wing-spot test in which we observe mutant wing hair colonies (spots) on the wings of adult flies obtained from the treated larvae. The induction of mutation was highest by irradiation at 310 nm and decreased as the wavelength became longer. Neither the 380 nor the 400 nm light was mutagenic. Excision repair is known to protect cells from UV damage. In the excision-repair–deficient Drosophila, the mutagenic response induced by 310 nm irradiation was 24-fold higher than that of the wild-type (7.2 ± 1.5 spots/wing/kJ vs 0.3 ± 0.08 spots/wing/kJ), and at 320 nm the difference of the response was 14-fold (0.21 vs 0.015 ± 0.005). In the case of irradiation at 330 and 340 nm the difference of the response was only two-fold (at 330 nm, 6.9 ± 2.9 × 10⁻³ vs 3.1 ± 1.1 × 10⁻³ spots/wing/kJ; at 340 nm, 3.5 ± 0.9 × 10⁻³ vs 2.0 ± 0.7 × 10⁻³). These results suggest that the lesion caused in the larvae by 320 nm irradiation may be similar to the damage induced by 310 nm and that the lights of 330 and 340 nm may induce damage different from the lesions induced by shorter-wavelength lights.

For facilitating photochemical and toxicological studies an ex vivo skin model was developed in our laboratory using skin from domestic pigs. The model comprised the use of a complete skin piece, including the dermis and stratum corneum, of bigger areas to make future topical applications easier. Fully differentiated skin explants (5 × 50 mm, thickness 5 mm) were irradiated with ultraviolet B (UVB; 1–10 kJ/m²; 6 W/m²). Directly thereafter they were brought in culture (Dulbeccos modified Eagles medium containing hydrocortisone; air/liquid interface) for a maximum of 144 h. In nonirradiated skin explants, signs of tissue degeneration were observed after 48 h in culture (hematoxylin and eosin, light microscope). However, keratinocytes, isolated enzymatically (thermolysin and trypsin) at different time intervals in culture from nonirradiated skin explants showed negligible loss in viability (trypan blue exclusion) and increased apoptosis (terminal deoxynucleotidyl transferase–mediated deoxy uridine triphosphatase nick end labeling assay) for up to 72 h. Explants irradiated with a single dose of UVB showed a clear and reproducible dose- and time-dependent tissue degeneration, loss in keratinocyte viability and increase in apoptosis compared with nonirradiated explants at the same time interval. In conclusion, the presently designed ex vivo pig skin model can be a useful and cheap tool for future investigations of short-term UV-induced effects in combination with phototoxic and photoprotective compounds.

The effects of short-term exposure to ultraviolet B (UVB) radiation on lymphocyte-related parameters were studied under controlled laboratory conditions using roach (Rutilus rutilus), a cyprinid teleost, as the model fish. In vitro lymphoproliferative responses stimulated with a T-cell–specific mitogen, concanavalin A (ConA), or a B-cell–specific activator, lipopolysaccharide (LPS), were decreased in exposed fish. Also nonstimulated proliferation was lower than in unexposed fish. ConA-activated responses returned to normal levels within 7 days after exposure, but LPS-activated responses were reduced throughout the 14 day follow-up. The capability of UVB-exposed fish to produce an antibody response was studied by intraperitoneal immunization with bovine γ-globulin (BGG). The concentration of anti-BGG antibodies in plasma as well as the number of anti-BGG–specific antibody-secreting cells in the spleen or blood were not decreased in fish exposed either to a single dose of UVB prior to immunization, or to single dose of UVB prior to immunization followed by three additional doses after immunization. Immunoglobulin M (IgM) production, when assayed as plasma IgM level or as the number of IgM-secreting cells in the spleen or blood, was not suppressed after exposure to UVB irradiation. These results indicate that a single dose of UVB or short-term exposure to UVB irradiation has no negative effects on IgM production or reactivity against antigen administered via the intraperitoneal route. However, the suppression of in vitro lymphoproliferative responses suggest that exposure to UVB has the potential to interfere with lymphocyte-related functions in fish.

Translocation from the outer to the inner membrane leaflet (flip) of phospholipids after ultraviolet A (UVA) irradiation was investigated in Chinese hamster ovary cells. Fluorescent 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl]-sn-glycero-3-phosphoserine (NBD-labeled phosphatidylserine [NBD-PS]) was used to assay transbilayer lipid movement. A marked increase in flip of NBD-PS was observed immediately after low-dose UVA irradiation which was not lethal and returned to the basal level after 6 h. UVA-induced flip was not attributed to the increase of permeability by UVA irradiation because cells that were negative for staining with propidium iodide also showed increased flip of NBD-PS. Furthermore, the enhancement was independent of adenosine 5′-triphosphate, demonstrating the lack of involvement of phospholipid translocase. Marked increases were also observed in flip of both NBD-phosphatidylethanolamine and NBD-phosphatidylcholine immediately after UVA irradiation, showing that the increase was independent on the head groups of phospholipids. These findings indicated that UVA changes the flip–flop of phospholipids and that the cell membrane is a molecular and cellular target of UVA.

Relatively little is known about the immunosuppression induced in mice which have received cutaneous photodynamic therapy (PDT). Consequently, experiments were undertaken using mice which received dorsal PDT using Photofrin^® as the photosensitizer in an attempt to characterize the overall nature of the immunosuppression. Photoirradiation of mice at various times after injection indicated there was no correlation between photosensitivity and immunosuppression. The suppression was found to be adoptively transferable and antigen specific suggesting the generation of suppressor cells. Selective cell depletions prior to adoptive transfer indicated a CD4 T cell to be responsible for the immunosuppression. Interestingly, using allogeneic spleen cells, no effect on the delayed type hypersensitivity (DTH) response was found. The results indicate that the suppression induced by cutaneous PDT, with the exception of the lack of DTH suppression, is similar to that induced by UVB irradiation but unlike that reported using laser PDT of the peritoneal cavity. This suggests that not only the type of photoirradiation but also the site of photoirradiation might determine the character of the induced immunosuppression.

We have previously reported the formation of wrinkles, a decrease in skin elasticity and a loss in the linearity of dermal elastic fibers in rat hind limb skin irradiated with ultraviolet radiation in wavelength ranging 290–320 nm (UVB) at a suberythemal dose for 6 weeks. Estrogens are considered effective in preventing photoaging in postmenopausal females, but the role of estrogen in the skin remains unclear. In this study we have evaluated the influence of short-term chronic UVB irradiation at a suberythemal dose on the skin of ovariectomized rats. An ovariectomy or a sham operation was performed on each 3 week–old female Sprague-Dawley rat. Starting 1 week after the operation the hind limb skin of each rat was irradiated with UVB at a suberythemal dose (130 mJ/cm²) three times a week for 3 or 6 weeks. Decreases in elasticity and wrinkle formation in the skins of ovariectomized animals were induced more quickly than in the skins of sham-operated animals following UVB irradiation. The linearity of elastic fibers in the ovariectomy group decreased significantly compared with the sham-operation group, but erythema in the ovariectomy group was induced more readily than in the sham-operation group following UVB irradiation. These findings suggest that decreases in the estrogen levels after ovariectomy accelerate photoaging in terms of the morphology and physical properties of the skin surface and the three-dimensional structure of elastic fibers.

Photosensitivity is an important characteristic feature of several forms of lupus erythematosus (LE), and induction of skin lesions by UV-A and UV-B irradiation has been proved to be an optimal model for evaluating light sensitivity in patients with this disease. Because lupus erythematosus tumidus (LET) has rarely been documented in the literature and is often difficult to differentiate from other photodermatoses such as polymorphous light eruption, we performed photoprovocation tests in 60 patients with LET according to a standardized protocol. Areas of uninvolved skin on the upper back were irradiated with single doses of UV-A (100 J/cm²) and/or UV-B (1.5 minimal erythema dose) daily for three consecutive days. Interestingly, patients with LET are more photosensitive than those with subacute cutaneous lupus erythematosus, and in our study experimental phototesting revealed characteristic skin lesions in 43 patients (72%). Because of the latency period in developing positive phototest reactions, it might be difficult for these patients to link sun exposure with their skin lesions. Furthermore, our data revealed a positive correlation of antinuclear antibodies and positive provocative phototest reactions in these patients as seen for other forms of LE. In conclusion, the high incidence of positive phototest reactions in correlation with the clinical findings, history of photosensitivity and antinuclear antibodies enable the classification of LET as the most photosensitive type of LE.

These studies addressed the hypothesis that UV radiation (UVR) could affect immune responses in mice infected with Borrelia burgdorferi. Immunity against the Lyme spirochete B. burgdorferi was studied in a murine model of UV-induced immune suppression. Borrelia-specific cellular and humoral responses were examined following immunosuppressive doses of UVR. Low-passage Borrelia were injected intradermally at the base of the tail following irradiation. At various time points after infection the blood was cultured for the presence of Borrelia and the serum analyzed for Borrelia-specific antibodies. Two weeks after infection one hind-limb joint was cultured for the presence of spirochetes and the contralateral joint was examined histologically for arthritis formation. The results demonstrated that UV irradiation, administered at the site of infection or at a distant site, suppressed Borrelia-specific cellular and humoral responses in infected mice. Suppression of delayed-type hypersensitivity and antibody responses to UV was abrogated by administration of anti–interleukin (IL)-10 after UV irradiation. In addition, UV irradiation altered the dissemination pattern of the bacteria from the skin into the blood and exacerbated arthritis when compared with unirradiated controls. From these studies we concluded that UV irradiation can modulate the immune response to Borrelia and exacerbate the subsequent arthritic component of Lyme disease in mice. Furthermore, our studies suggest that IL-10 is in part responsible for the suppression of both cellular and humoral responses in addition to playing a role in the development of Lyme arthritis.

A low level of chemilumnescence by hemoglobin (Hb) was detected in the reaction with H₂O₂ and hydrogen donors such as gallic acid and catechins. The photon intensity was affected by the ferric state of Hb (methemoglobin > oxyhemoglobin), and was roughly correlated with the radical-scavenging potential of catechins. We hypothesized the reversible activation reaction of Hb as the chemiluminescence mechanism of the H₂O₂/gallic acid/Hb system. It is indicated that the oxidized-Hb (Hb-OOH) formation was a chemiluminescence-rate–determining step and one-electron reduction by a hydrogen donor of the compound-I–type intermediate ([^·XFe^IV] = O) proved a chemiluminescence-specificity–determining step. Spectral analysis showed that the photon emission from the H₂O₂/gallic acid/Hb system was produced without singlet oxygen generation. The concentration dependence of photon intensity suggests a high consumption ratio of H₂O₂ leading to protection from H₂O₂ toxicity. Albumin was defined as a hydrogen donor by the isolation of chemiluminescent substance in plasma using this chemiluminescence system.

The potential of ketocarotenoids to protect the photosynthetic apparatus from damage caused by excess light and UV-B radiation was assessed. Therefore, the cyanobacterium Synechococcus was transformed with a foreign β-carotene ketolase gene under a strong promoter leading to the accumulation of canthaxanthin. This diketo carotenoid is absent in the original strain. Most of the newly formed canthaxanthin was located in the thylakoid membranes. The endogenous β-carotene hydroxylase was unable to interact with the ketolase. Therefore, only traces of astaxanthin were found. The transformant was treated with strong light (500 or 1200 μmol m⁻² s⁻¹) and with UV-B radiation. In contrast to a nontransformed strain the overall photosynthesis, measured as oxygen evolution, was protected from inhibition by light of 500 μmol m⁻² s⁻¹ and UV-B radiation of 6.8 W m⁻². Furthermore, degradation in the light of chlorophyll and carotenoids at an irradiance of 1200 μmol m⁻² s⁻¹, which was substantial in the nontransformed control, was prevented. These results indicate that in situ canthaxanthin, which is formed at the expense of zeaxanthin and replaces this hydroxy carotenoid within the photosynthetic apparatus, is a better protectant against solar radiation. These findings are discussed on the basis of the in vitro properties such as inactivating peroxyl radicals, quenching of singlet oxygen and oxidation stability of these different carotenoid structures.

C-Phycocyanin (PC) trimers associated with linker polypeptides were isolated from the phycobilisome (PBS) rods of Synechococcus sp. PCC 7002. L_X^Y refers to a linker polypeptide (L) having an apparent mass of Y kDa, located at position X in the phycobilisome where X can be R (rod), C (core) or RC (rod–core junction). Measurements of the absorption, fluorescence and excitation anisotropy of PC trimer, PC·L_R^32.3 and PC·L_RC^28.5 complexes document the spectroscopic modulation of each linker polypeptide on the PC chromophores. The difference spectra between the PC trimer and the PC–linker complexes show that although the effect induced by the linker polypeptides is qualitatively similar in behavior, the extent of the modulation is greater in PC·L_RC^28.5. Measurements taken at 77 K show that a red-wavelength component of the PC trimer absorption–fluorescence spectra is the target of the linker's influence and that this component is altered to a greater extent by L_RC^28.5. In addition the 77 K absorbance of the PC trimer resolves band features that are consistent with an excitonic coupling interaction between neighboring α84 and β84 chromophores. These band features are also evident in the absorbance of PC·L_R^32.3 but are absent in PC·L_RC^28.5 indicating that L_RC^28.5 may be perturbing the coupling interaction established in the PC trimer α84–β84 chromophore pairs. Structurally, the linker polypeptide should disrupt the C₃ symmetry in the central cavity of the associated phycobiliprotein and this asymmetric interaction should serve to guide the transfer of excitation energy along PBS rods toward the core elements.

The effect of metal cation binding on bacteriorhodopsin (bR) in purple membrane has been examined using in situ attenuated total reflection-Fourier transform infrared difference spectroscopy in aqueous media. It is known that adding metal cations to deionized bR regenerates the purple state from its blue state and recovers the proton pump function. During this process, infrared spectral changes in the frequency region of 1800–1000 cm⁻¹ are monitored. The results reveal that metal cation binding affects the protein conformation, the retinal isomeric composition as well as lipid head groups. It is also observed that metal cation binding induces conformational changes in the α_I-helix region of bR, converting the portion of its α_I-helical domain into β-turn or disordered coil. In addition, the influence of Ho³ binding on the protein and lipid is observed to be larger than that of Ca² . These results suggest that some of the metal cation binding sites are on the membrane lipid domain, while others could be on the intrahelical domain or interhelical loops where the Asp and Glu are located (binding with their COO⁻ groups). Our results also suggest that the removal of the C-terminal of bR increase the accessibility of the binding site of metal cations, which affects protein conformational structure. All these observations are discussed in terms of the two proposals given in the literature regarding the metal cation binding sites.