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.     

THE QUENCHING OF EXCITED STATES OF HYPOCRELLIN A BY RARE EARTH IONS

The quenching processes of prompt and delayed fluorescence of hypocrellin A by rareearth ions were investigated. It is demonstrated that the prompt fluorescence of HA can bequenched efficiently by RE in methanol via a static quenching process including a groundstate complex formation. The thermally induced delayed fluorescence of HA can also bequenched effectively by complex formed, which quenching is of dynamic quenching behavior.The fluorescence quenching of HA in various micellar systems which protect the quenchingby the compartmentalization of micelles is discussed as well.     

螺吡喃激发态的反应活性

研究了6′-硝基-1,3,3-三甲基吲(口乃木)苯骈螺吡喃,Ⅰ,和6′-硝基-1-苯基-3,3-二甲基吲(口乃木)苯骈螺吡喃,Ⅱ,的光化学,Ⅰ的直接光解和敏化光解均得到份菁结构的光解产物Ⅲ,量子产率分别是0.42和0.32.Ⅱ直接光解时生成相应的有色光解产物Ⅳ,量子产率是0.40,氮杂蒽酮为敏化剂的敏化光解量子产率是0.58,无疑地,单线态和三线态均参予螺吡喃的发色反应,用这些结果和折算关系式得到,在Ⅰ的直接光解中,Ⅲ主要来自单线态,而在Ⅱ的直接光解中,Ⅳ主要来自三线态.从Ⅰ或Ⅱ的敏化联乙酰燐光实验中求得三线态参数.它们是:φ_ISC^Ⅰ＝0.11,φ_ISC^Ⅱ＝0.48;³K_dt^Ⅰ＝3×10⁴秒^-1,³K_dt^Ⅱ＝9×10⁴秒^-1,类似地,³K_r^Ⅰ＝0.12×10⁴秒^-1,³K_r^Ⅱ＝2.52×10⁴秒^-1,这样,³φ_r^Ⅰ值较小是由于³φ_ISC^Ⅰ值较小,而三线态寿命随结构变动不大.最后,取苯乙烯为模型,用它的电子能量与乙烯双键扭曲角度关系图推断出,来自单线态的双离子具有螺环构型,而来自三线态者具有平面状构型.     

EXCITED STATES OF FLAVINE COENZYMES-TV. KINETICS OF THE PHOTOREDUCTION OF LUMIFLAVINE BY METHIONINE

Abstract— Kinetics of fluorescence quenching and photoreduction of lumiflavine by methionine in anaerobic aqueous solution show that the flavine reacts in the excited triplet state at a diffusion controlled rate. Individual rate constants and activation energies are determined for the process.     

THE GENERATION OF HYDROGEN PEROXIDE BY THE UVA IRRADIATION OF HUMAN LENS PROTEINS

Abstract— The water-insoluble proteins from aged human lens are known to contain protein-bound chromophores that act as UVA sensitizers. The irradiation of a sonication-solubilized, water-insoluble fraction from human lenses (55–75 years) with UVA light (1.5 kj/cm², λ > 338 nm) caused an oxygen-dependent photolysis of tryptophan, not seen when either α-crystallin or lysozyme were irradiated. The suggested requirement for active oxygen species was consistent with a linear increase in hydrogen peroxide formation, which was also observed. A final concentration of 55 µM H₂O₂ was attained, with no H₂0₂ being detected in either dark-incubated controls or in irradiated samples of native proteins. The UVA-dependent H₂O₂ formation was increased 50% by superoxide dismutase (SOD) and abolished by catalase, arguing for the initial generation of superoxide anion. A linear photolysis of histidine and tryptophan was also seen; however, the addition of SOD or SOD and catalase had no effect on the photolytic destruction of either amino acid. Superoxide dismutase increased the oxidation of protein SH groups implicating H₂O₂, but SOD and catalase caused a decrease in SH oxidation only at later time periods. The direct addition of H₂O₂ to a water-insoluble sonicate supernatant fraction caused only a slight oxidation of SH groups, but this was increased four- to eight-fold when the protein was denatured in 4.0 M guanidine hydrochloride. Overall, the data suggest a UVA-dependent oxidation of protein SH groups via H₂O₂ generated within the large protein aggregates of the water-insoluble fraction. These data also provide a mechanism for oxidation of the sulfur-containing amino acids in vivo—a process that is known to accompany the formation of age-onset cataracts.     

ENZYMATIC GENERATION OF ELECTRONICALLY EXCITED STATES BY ELECTRON TRANSFER

Abstract— The catechol oxidase promoted oxidation of catechols produces electronically excited states readily detected by chlorophyll sensitized fluorescence. Both the chemiexcitation step and the transfer step are efficient. The use of chlorophyll has confirmed that the peroxidase catalyzed oxidation of dihydroxyfumarate also generates electronically excited species. It is concluded that efficient enzymatic generation of electronic energy can occur not only through the dioxetane/dioxetanone route, but also via electron transfer processes.     

ELECTRONICALLY EXCITED STATES IN MICROSOMAL MEMBRANES: USE OF CHLOROPHYLL-a AS AN INDICATOR OF TRIPLET CARBONYLS

Abstract —Chlorophyll- a enhances the photoemission from the microsomeltert-butyl hydroperoxide system without affecting the rates of O² uptake and lipid peroxidation. This photoemission, due to energy transfer from triplet carbonyls to microsome-bound chlorophyll- a , shows that microsomal membranes produce a significant amount of excited triplet carbonyls during lipid peroxidation.     

EXCITED STATES OF FLAVINES – V. THE QUENCHING OF FLAVINE LUMINESCENCE BY INDOLES

Abstract— The quenching of the fluorescence of flavines by indoles in aqueous solution has been studied as a function of concentration, viscosity and temperature. The quenching shows a negative temperature dependence, and is in part diffusion controlled. Static and dynamic processes both contribute to quenching.
In the presence of alcohol or denaturants static quenching is inhibited and dynamic quenching supervenes. The effect of indoles on the flavine fluorescence lifetimes and phosphorescence characteristics is consistent with the quenching mechanism proposed.     

PORPHYRIN PHOTOSENSITIZATION OF PROTEINS IN CELL MEMBRANES AS STUDIED BY SPIN-LABELLING AND BY QUANTIFICATION OF DTNB-REACTIVE SH-GROUPS

Abstract— Human cells of the line NHIK 3025 were exposed to hematoporphyrin derivative (Hpd) and light and analysed with respect to; (i) the mobility of membrane proteins as determined by electron spin resonance measurements of a protein-bound spin label, (ii) fluorescence excitation spectra, (iii) relative number of DTNB-reactive SH-groups on their surface and in sonicated cell homogenates, (iv) survival, and (v) morphologic appearance as seen by ordinary phase contrast microscopy. A significant fraction of the porphyrins bound to the outer cell membrane was in close contact with proteins. 5,5'-Dithiobis-2-nitrobenzoic acid reactive SH-groups on the outer cell membrane were very sensitive to the treatment with Hpd + light and were degraded according to non-exponential kinetics. When the cells were irradiated after spin labelling, the labelled proteins became less mobile during the irradiation, indicating protein cross linking. Irradiation before spinlabelling resulted in a selective degradation of low-mobility proteins.     

THE INTERACTION OF THE EXCITED STATES OF SAFRANINE T WITH ALIPHATIC AMINES IN ORGANIC SOLVENTS

Abstract—
The reactions of the excited states of safranine T with aliphatic amines have been studied in methanol and acetonitrile. Quenching of the singlet and triplet states occurs by different mechanisms. Whereas the former excited state is quenched by a charge-transfer mechanism, the triplet state is quenched through proton transfer from the excited dye to the amine. This process leads to the unprotonated form of the dye in the triplet state, which is later quenched by amines to form the corresponding semireduced species. The monoprotonated triplet also undergoes self-quenching in both solvents (k = 1.2 × 10⁸ M ^-1 s^-1).     

QUENCHING OF THE EXCITED STATES OF 2-PHENYLBENZOXAZOLE BY AZIDE ANION. FLUORESCENCE AND ESR STUDY

Electron spin resonance, spin-trapping and fluorescence techniques demonstrate that 2-phenylbenzoxazole (P) participates in photo-induced reactions with alcohols and electron donors like the azide ion. Irradiation of Pat 300 nm in deaerated ethanol produces ethoxyl and hydroxyethyl radicals which can be detected with the spin trap, 5,5-dimethyl-l-pyrroline-l-oxide (DMPO). However, irradiation of P in the presence of N^-₃ leads to the appearance of the azide radical, N^-₃, which also reacts with DMPO. Studies with the nitroxyl radical, 2,2,6,6-tetramethylpiperidine-l-oxyl (TEMPO), suggest that electron transfer from the azide anion to an excited state of P yields the semi-reduced sensitizer, P^-, which in turn reacts with TEMPO. The effect of sodium azide upon the fluorescence intensity and lifetime of P in aqueous ethanol has also been studied.     

THE PHOTOCHEMISTRY OF HUMAN RETINAL LIPOFUSCIN AS STUDIED BY EPR

Fluorescent material generated in the human retina accumulates within lipofuscin (HLF) granules of the retinal pigment epithelium (RPE) during aging. We have been investigating the possible light-induced contribution of these fluorophores to various diseases including age-related macular degeneration. Our studies have shown that some of the fluorescent components of HLF are products of the reaction of retinaldehyde with ethanolamine and that synthetic mixtures of this reaction can serve as a useful model for photophysical studies. Previous research by us has demonstrated that irradiation of either natural or synthetic lipofuscin resulted in the formation of a triplet state and possibly a free radical. Here EPR studies were performed to verify the formation of that radical. The UV irradiation of either synthetic or natural human retinal lipofuscin extracts in oxygen-free methanol led to the formation of a 5,5-dimethylpyrroline-N-oxide (DMPO) spin-trapped carbon-centered radical resulting from either hydrogen atom or electron abstraction from solvent molecules. In the presence of oxygen superoxide was formed, which was observed as a DMPO adduct. It is concluded that certain components of the chloroform-soluble fluorophores of human RPE lipofuscin granules and the fluorescent reaction products of retinaldehyde and ethanolamine are photophysically similar but not the same. Electron or hydrogen abstraction from a substrate by these fluorophores in vivo and the resulting radical products may contribute to the age-related decline of RPE function and blue light damage in the retina.     

LIGHT-INDUCED CONFORMATIONAL CHANGES IN CATTLE RHODOPSIN AS PROBED BY MEASUREMENTS OF THE INTEFFACE POTENTIAL

Abstract— A novel technique of capacitative coupling of oriented rhodopsin at a polar/apolar interface allows the time resolved investigation of conformational changes following a flash. Electric signals arise as a consequence of changes of the interface potential. A signal occurring within milliseconds behaves like the R₂-phase of the "early receptor potential" (= ERP). This response is interpreted as a conformational change of rhodopsin. No correlation of this signal is found to the spectroscopically defined metarhodopsin I-metarhodopsin II transition.
The temperature dependence of the conformational change coincides with the temperature dependence of the latency of the 'a-wave' of the electroretinogram, reported by Arden and Ikeda (1968). It is suggested that the command step of visual excitation is the conformational change and not one of the spectroscopically defined photolysis steps of rhodopsin.
Analysis of slower electrical signals following the fast response is in accordance with the model of a light-induced pore formation.     

THE PRIMARY REACTION IN THE PHOTOREDUCTION OF PROTOCHLOROPHYLLIDE MONITORED BY NANOSECOND FLUORESCENCE MEASUREMENTS

Abstract— Time resolved fluorescence measurements, carried out on protochlorophyllide reductase enriched membranes from oat ( Avena sativa ), are described. A fast (1 ns at 293 K) decaying fluorescence component is assigned to the photoactive NADPH-protochlorophyllide-enzyme complex, while a slower (5 ns) component is ascribed to non-photoactive protochlorophyllide. The results are interpreted in terms of a new fast primary step in the light requiring step of chlorophyll synthesis. The temperature dependence of the rate of this reaction has been studied by measuring the decay time of the fast fluorescence component at various temperatures from 77 to 293 K. Complete spectra of the kinetic fluorescence components have been measured at 293, 160 and 77 K.     

分子离子NO+激发态的势能函数

100公里以上的大气分子离子主要为NO~+。它的辐射特点及它与电子、原子或分子的相互作用,对于理解大气的化学过程具有特别重要的意义。为了研究这些过程,确定NO~+分子离子基态及其各个激发态的分子势能函数是非常重要的。精确的X~1Σ~+,A~1Ⅱ和a~3∑~+势能曲线已发表;基于光电子谱的研究发现了NO~+的其它激发态,但对于这些激发态的研究尤其是势能函数的研究不多。本文研究并导出NO~+的基态和10个激发态的势能函数。     

EXCITED STATES OF FLAVINE COENZYMES–II. ANAEROBIC OXIDATION OF AMINO ACIDS BY EXCITED RIBOFLAVINE DEVRIVATIVES

Abstract— Flavines in the excited state can effect anaerobic oxidation of amino acids to keto acid and ammonia. The rate of the process depends on the structure of the amino acid. These systems show similarities to the amino acid oxidases.     

ON THE ROLE OF FORBIDDEN LOW-LYING EXCITED STATES OF LIGHT-HARVESTING CAROTENOIDS IN ENERGY TRANSFER IN PHOTOSYNTHESIS*

Abstract—It is proposed that the low-lying. electric-dipole forbidden A_g excited electronic state recently discovered in β-carotene acts as the energy donor in Förster-type excitation transfer to antenna Chl molecules folbwing β-carotene's light-harvesting function in photosynthesis. The mechanism and conclusion should apply as well to all carotenoid accessory pigments.