Resonance hyper-Raman excitation profiles and two-photon states of a donor-acceptor substituted polyene

Resonance Raman and resonance hyper-Raman spectra and excitation profiles have been measured for a "push-pull" donor-acceptor substituted conjugated polyene bearing a julolidine donor group and a nitrophenyl acceptor group, in acetone at excitation wavelengths from 485 to 356 nm (two-photon wavelengths for the nonlinear spectra). These wavelengths span the strong visible to near-UV linear absorption spectrum, which appears to involve at least three different electronic transitions. The relative intensities of different vibrational bands vary considerably across the excitation spectrum, with the hyper-Raman spectra showing greater variation than the linear Raman. A previously derived theory of resonance hyper-Raman intensities is modified to include contributions from purely vibrational levels of the ground electronic state as intermediate states in the two-photon absorption process. These contributions are found to have only a slight effect on the hyper-Rayleigh intensities and profiles, but they significantly influence some of the hyper-Raman profiles. The absorption spectrum and the Raman, hyper-Rayleigh, and hyper-Raman excitation profiles are quantitatively simulated under the assumption that three excited electronic states contribute to the one- and two-photon absorption in this region. The transition centered near 400 nm is largely localized on the nitrophenyl group, while the transitions near 475 and 355 nm are more delocalized.     

ACTION SPECTRA FOR HYPHAL AGGREGATION,THE FIRST STAGE OF FRUITING,IN THE BASIDIOMYCETE Pleurotus ostreatus*

Abstract. Action spectra were determined for hyphal aggregation in Pleurotus ostreatus at wavelengths between 360 and 600 nm. The action spectrum for a 50% response had two maxima, one at 370 nm in the near-UV and the other a broad peak at 440–450 nm in the blue. Both were approximately of the same magnitude. A minimum was present at 400 nm and wavelengths greater than 530 nm invoked no response. Action spectra for a range of responses, 10–90%, were also determined which showed gradual changes in the peaks in the blue region. It was concluded that hyphal aggregation in P. ostreatus is under the control of a cryptochrome-like photoreceptor system.     

A monochromatic action spectrum for the photoinduction of the UV-absorbing mycosporine-like amino acid shinorine in the red alga Chondrus crispus

To determine the action spectrum for photoinduction of the ultraviolet (UV)-absorbing mycosporine-like amino acid shinorine, specimens of the marine red alga Chondrus crispus were irradiated with monochromatic light of various wavelengths using the Okazaki large spectrograph at the National Institute for Basic Biology, Okazaki, Japan. Fluence response curves were determined for the wavelengths between 280 and 750 nm, by irradiating the algae with monochromatic light for 10 h, followed by 4 h of 25 micromol m(-2) s(-1) photosynthetically active radiation and 10 h darkness. Samples were taken after the second exposure interval. A linear correlation between fluence rate and accumulated shinorine concentration was detected for wavelengths between 350 and 490 nm in the fluence rate range of 20-30 micromol m(-2) s(-1), whereas there was no induction above 490 nm. Below 350 nm a decline in shinorine concentration could be observed at fluence rates above 30 micromol m(-2) s(-1), probably due to an inhibition of photosynthetic activity and a subsequent impairment of shinorine biosynthesis. The constructed action spectrum indicated that the photoreceptors mediating shinorine photoinduction might be an unidentified UV-A-type photoreceptor with absorption peaks at 320, 340 and 400 nm.     

LIGHT-INDUCED SYNTHESIS OF ANTHOCYANIN IN CARROT CELLS IN SUSPENSION—IV. THE ACTION SPECTRUM

Abstract— Using carrot cell suspension in 2,4-dichlorophenoxyacetic acid (2,4-D)-depleted culture medium, fluence-response curves for the formation of anthocyanin were determined at various wavelengths from 250 to 800 nm. In the fluence-response curves at wavelengths between 260 and 330 nm, the response showed a sharp fluence-dependent increase after the fluence exceeded threshold level at the respective wavelength. Such a sharp increase in response was not observed by light at 450 nm or longer wavelengths, although the response obtained by higher fluence of such light was always higher than that in the dark control. Action spectra determined at the sharp increasing phase of the response showed the single peak at 280 nm which equals the absorption maximum of UV-B photoreceptor.
Although red (R)-light alone had a minor effect on anthocyanin accumulation, it modulated the action of UV-B light. That is, when carrot cells were irradiated with R-light either before or after UV-B irradiation, anthocyanin formation was greatly enhanced above the level enhanced by UV-B light alone. The most effective wavelength for this enhancement was 660 nm. The effect of R-light on the anthocyanin formation of the UV-B irradiated cells was reversed by immediately following it with far-red light, suggesting the involvement of phytochrome in the R-effect.     

Resonance hyper-Raman spectra of zinc phthalocyanine

Hyper-Raman spectra were obtained for zinc phthalocyanine in a dilute pyridine solution at excitation wavelengths that are two-photon resonant with the one-photon-allowed B band (360-380 nm) as well as with the two-photon absorption near 440 nm reported by Drobizhev et al. ( J. Chem. Phys. 2006, 124, 224701 ). In both regions, the hyper-Raman spectra were very different from the linear resonance Raman spectra at the corresponding excitation frequencies. While the resonance Raman spectra show only g symmetry modes, almost all of the hyper-Raman frequencies can be assigned as fundamentals of E u symmetry that also are observed in the infrared absorption spectrum or E u symmetry combination bands. These results contrast sharply with previous observations of highly noncentrosymmetric push-pull conjugated molecules and are consistent with a structure for phthalocyanine in solution that is centrosymmetric or nearly so. The hyper-Raman spectra show different intensity patterns in the two excitation regions, consistent with different Franck-Condon and/or vibronic coupling matrix elements for the different resonant states.     

A novel prokaryotic UVB photoreceptor in the cyanobacterium Chlorogloeopsis PCC 6912

We present evidence for the presence and nature of a UVB-specific photoreceptor in the cyanobacterium Chlorogloeopsis PCC 6912. The photoreceptor mediates at least the photosensory induction of mycosporine-like amino acid (MAA) synthesis. Because MAA synthesis in this organism can also be induced under salt stress, we could distinguish between the photosensory and the purely biochemical requirements of MAA synthesis. Neither visible light nor UV radiation was necessary for the biosynthetic process, thus indicating that the UVB (280-320 nm) dependence of biosynthesis is based on a UV photosensory capacity of the organism. An action spectrum of the MAA synthesis showed a distinct peak at 310 nm tailing down into the UVA (320-400 nm) region with no detected activity above 340 nm. We found that radiation below 300 nm caused significant inhibition of synthesis of MAAs indicating that the action spectrum at these wavelengths may not have been satisfactorily resolved. We propose that a pterin is a good candidate for a photoreceptor chromophore as (1) reduced pterins present absorption spectra congruent with the action spectrum obtained; and (2) an inhibitor of the biosynthetic pathway of pterins and an antagonist of excited states of pterins, both depressed the photosensory efficiency of induction but not its chemosensory efficiency.     

Ultraviolet action spectra for DNA dimer induction, lethality, and mutagenesis in Escherichia coli with emphasis on the UVB region

Abstract —Ultraviolet (UV) action spectra were obtained for lethality and mutagenesis (reversion to tryptophan independence) in Escherichia coli WP2s for wavelengths 254–405 nm with detailed analysis in the UVB region (290–320 nm). Parallel chemical assay yields of pyrimidine dimers in DNA of E. coli RT4 were determined at the same wavelengths. Spectral regions isolated from a Xe arc and resonance lines from a high-pressure Hg-Xe arc lamp were both used for irradiation. In all cases, precise energy distributions throughout the isolated Xe bands regions were defined.
Lethality, mutagenesis, and dimer induction all decreased in efficiency in a similar fashion as the wavelengths of the radiation increased. Between 300 and 320 nm, all characteristics measured showed differences of about two and a half orders of magnitude. Between these wavelengths, the values of the three end points used either coincide with or parallel the absorption spectrum of DNA. The mutagenesis action spectrum coincides closely with the absorption spectrum of DNA. The lethality spectrum is closely parallel to the mutagenicity spectrum; the points, however, consistently occur at about 2 nm longer wavelengths. A calculation derived from the slope of the UVB spectra reveals that a 1-nm shift of the solar UV spectrum to shorter wavelengths would result in a 35% increase in its mutagenic potential. At 325 nm, both biological action spectra show sharp decreases in slope. In addition, above 325 nm the spectra for lethality. mutagenicity, and dimer formation diverge sharply; lethalities at these UVA wavelengths were approximately tenfold greater relative to mutagenicity than at shorter wavelengths. The relative yield of dimer formation by 365 nm radiation is intermediate between the yields for lethality and mutagenesis.     

In vivo absorption spectra of the two stable states of the Euglena photoreceptor photocycle

Euglena gracilis possesses a simple but sophisticated light detecting system, consisting of an eyespot formed by carotenoids globules and a photoreceptor. The photoreceptor of Euglena is characterized by optical bistability, with two stable states. In order to provide important and discriminating information on the series of structural changes that Euglena photoreceptive protein(s) undergoes inside the photoreceptor in response to light, we measured the in vivo absorption spectra of the two stable states A and B of photoreceptor photocycle. Data were collected using two different devices, i.e. a microspectrophotometer and a digital microscope. Our results show that the photocycle and the absorption spectra of the photoreceptor possess strong spectroscopic similarities with a rhodopsin-like protein. Moreover, the analysis of the absorption spectra of the two stable states of the photoreceptor and the absorption spectrum of the eyespot suggests an intriguing hypothesis for the orientation of microalgae toward light.     

双波长分光光度法同时测定水杨醛和苯酚

利用水杨醛、苯酚的紫外吸收光谱特征及水杨醛在271 nm波长处有一个等吸收点,提出了同时测定水杨醛和苯酚的双波长分光光度法。选择328 nm和271 nm为测定波长,以其回归方程为基础建立了联立方程组,C水杨醛=31.31A328 nm+0.344;C苯酚=44.53A271 nm-1.866A328 nm-0.136,求得组分浓度与吸光度之间的关系,浓度在0.005~0.030 g.L-1范围内符合朗伯-比耳定律。     

Resonance hyper-Raman excitation profiles of a donor-acceptor substituted distyrylbenzene: one-photon and two-photon states

Resonance Raman and resonance hyper-Raman spectra of the "push-pull" conjugated molecule 1-(4'-dihexylaminostyryl)-4-(4"-nitrostyryl)benzene in acetone have been measured at excitation wavelengths from 485 to 356 nm (two-photon wavelengths for the nonlinear spectra), resonant with the first two bands in the linear absorption spectrum. The theory of resonance hyper-Raman scattering intensities is developed and simplified using assumptions appropriate for intramolecular charge-transfer transitions of large molecules in solution. The absorption spectrum and the Raman, hyper-Rayleigh, and hyper-Raman excitation profiles, all in absolute intensity units, are quantitatively simulated to probe the structures and the one- and two-photon transition strengths of the two lowest-energy allowed electronic transitions. All four spectroscopic observables are reasonably well reproduced with a single set of excited-state parameters. The two lowest-energy, one-photon allowed electronic transitions have fairly comparable one-photon and two-photon transition strengths, but the higher-energy transition is largely localized on the nitrophenyl group while the lower-energy transition is more delocalized.     

IMMEDIATE PIGMENT DARKENING: VISUAL AND REFLECTANCE SPECTROPHOTOMETRIC ANALYSIS OF ACTION SPECTRUM

Immediate pigment darkening (IPD) occurs in human skin upon exposure to ultraviolet-A and visible radiation. The spectral changes that occur during IPD were measured with a rapid scanning reflectance spectrophotometer (RS) which employs optical fiber bundles for delivery and detection of light between 400 and 750 nm. The radiation dose dependence and wavelength dependence (334-549 nm irradiation) of IPD were studied by both the classical visual grading method and by spectrophotometric scoring using the RS system. The spectral changes that occur at long wavelengths with IPD mimic the natural absorption spectrum of melanin. Therefore, the IPD was scored in terms of the apparent change in melanin optical density, using the method Kollias and Baqer [Photochem. Photobiol. 43, 49-54 (1986)], based on reflectance in the 620-720 nm range. The nonlinearity of the visual grading method is demonstrated. The degree of IPD is first-order with respect to delivered dose and saturates after high doses. The maximum amount of IPD attained at saturation is greater for shorter wavelengths. Extrapolation of the reflectance data suggests the longest wavelength capable of eliciting IPD is about 470 nm.     

Initial excited-state structural dynamics of 2'-deoxyguanosine determined via UV resonance Raman spectroscopy

The resonance Raman spectra of 2'-deoxyguanosine, a DNA nucleoside, were measured in aqueous solution at wavelengths throughout its 260 nm absorption band. Self-consistent analysis of the resulting resonance Raman excitation profiles and absorption spectrum using a time-dependent wave packet formalism with two electronic states yielded the initial excited-state structural dynamics in both states. The vibrational modes containing the N(7)═C(8) stretching and C(8)-H bending internal coordinates were found to exhibit significant initial structural dynamics upon photoexcitation to either state and are coincident with the photochemical reaction coordinate involving the formation of the 2'-deoxyguanosine cation radical.     

SPECTRAL PROPERTIES OF THE RHODOPSIN-SYSTEM OF THE CRAYFISH Astacus leptodactylus

Abstract— The absorption spectra of the membrane-bound and of the digitonin-solubilized visual pigment of crayfish Astacus leptodactylus were investigated by conventional spectrophotometry. A method was developed to isolate purified rhabdoms almost entirely free from screening pigments from a single retina. The quantity of isolated and purified rhabdoms from a single retina was sufficient to measure the absorption spectra of the visual pigment.
The absorption spectra of the chromoprotein system (R and M) show that both the membrane-bound and the digitonin-solubilized visual pigment isomers are stable at 0°C and pH 7.0. Rhodopsin and metarhodopsin are photoreversible under these conditions without any light-induced denaturation. The difference spectra for the chromoprotein isomers and those of different photostationary states yield maximal values for ΔE at 570 and 485 nm.
At neutral pH, 0°C, Λ_max of rhodopsin is 530 nm. Irradiation with light of Λ= 630 to 640 nm isomerizes rhodopsin nearly quantitatively to metarhodopsin with Λ_max, of 500 nm. The molar extinction coefficient of metarhodopsin is greater than that of rhodopsin by a factor of ˜ 1.41. each measured at its respective Λ_max Metarhodopsin can be isomerized to rhodopsin by irradiating at Λ > 630 nm. As the absorption spectra of the two chromoprotein isomers overlap, only part of the metarhodopsin can be reversed to rhodopsin. The maximal photoreversion can be achieved by irradiating at 460 nm. The stability of the digitonin-solubilized chromoprotein is remarkably dependent on temperature. Warming the digitonin extract of rhabdoms from 0 to 20 or 30°C caused a shift of the rhodopsin spectrum to shorter wavelengths (Λ_max= 485 nm) accompanied by a decrease of E_Λmax by about 30%.     

Photooxidation of PVC

Pure and peroxided PVC films were irradiated under monochromatic ultraviolet light between 300 and 400 nm. The degradation state of PVC is measured by means of carbonyl absorption in the infrared spectra and polyene content by UV spectroscopy. The initial rate of carbonyl formation depends on the wavelengths and oxidized impurities content. Peroxided PVC is oxidized faster than pure PVC with wavelengths above 320 nm. Two hazardous ranges of wavelengths have been detected: 300–320 and 350–370 nm. Good protection of PVC against UV radiation can be assured by the exclusion of wavelengths under 380 nm.     

Resonance Raman intensity analysis of the A band short-time photochemical dynamics of 4,5-ethylenedithio-1,3-dithiole-2-thione

Resonance Raman spectra (RRs) for 4,5-ethylenedithio-1,3-dithiole-2-thione (EDDT) were obtained with 397.9 and 416 nm excitation wavelengths, and density functional calculations were performed to elucidate the electronic transitions and the RRs of EDDT in chloroform solvent. The RRs indicate that the Franck-Condon region photodynamics have multidimensional character with nuclear motion predominantly along the C(4)═C(5) stretch and the C(4)═C(5) twist out-of-plane. Resonance Raman cross-sections of A-band absorption have been obtained for the vibrational modes of EDDT with its excitation frequencies spanning the 408 nm. Resonance Raman intensity analysis of the resulting RRs excitation profiles and absorption spectrum using a time-dependent wave packet formalism yields mode-specific nuclear displacement and vibrational reorganizational energies. The intensity analysis results for EDDT were compared to previously reported results for dimethyl 1,3-dithiole-2-thione-4,5-dicarboxylate (DDTD), which shows that the additional six-member heterocycle of EDDT strongly affects the reorganizational energy and energy participation. The authors briefly discuss the differences and similarities of the spectra in terms of molecular symmetry and electron density.     

Excited-state structural dynamics of cytosine from resonance Raman spectroscopy

Cytosine, a nucleobase found in both DNA and RNA, is known to form photoproducts upon UV irradiation, damaging the nucleic acids and leading to cancer and other diseases. To determine the molecular mechanism by which these photoproducts occur, we have measured the resonance Raman spectra of cytosine at wavelengths throughout its 267 nm absorption band. Self-consistent analysis of the resulting resonance Raman excitation profiles and absorption spectrum using a time-dependent wave packet formalism yields both the excited-state structural changes and electronic parameters. From this analysis, we have been able to determine that, at most, 31% of the reorganization energy upon excitation is directed along photochemically relevant modes.     

Excited-state structural dynamics of 5-fluorouracil

5-Fluorouracil is an analogue of thymine and uracil, nucleobases found in DNA and RNA, respectively. The photochemistry of thymine is significant; UV-induced photoproducts of thymine in DNA lead to skin cancer and other diseases. In previous work, we have suggested that the differences in the excited-state structural dynamics of thymine and uracil arise from the methyl group in thymine acting as a mass barrier, localizing the vibrations at the photochemical active site. To further test this hypothesis, we have measured the resonance Raman spectra of 5-fluorouracil at wavelengths throughout its 267 nm absorption band. The spectra of 5-fluorouracil and thymine are very similar. Self-consistent analysis of the resulting resonance Raman excitation profiles and absorption spectrum using a time-dependent wave packet formalism suggests that, at most, 81% of the reorganization energy upon excitation is directed along photochemically relevant modes. This compares well with what was found for thymine, supporting the mass barrier hypothesis.     

ACTION SPECTRA FOR INHIBITION OF HYPOCOTYL GROWTH OF WILD-TYPE PLANTS AND OF THE hy2 LONG-HYPOCOTYL MUTANT OF Arabidopsis thaliana L.

Abstract— An analysis was made by action spectroscopy, using the Okazaki Large Spectrograph, of the inhibition of hypocotyl elongation of wild-type plants and the hy2 mutant of Arabidopsis thaliana. Two day old etiolated seedlings were irradiated for 8 h with monochromatic light and left in the dark for 16 h before measurement of hypocotyl length. Spectrophotometric measurement showed that levels of phytochrome in the etiolated tissue of the hy2 mutant were less than 9% of those in the wild type. The action spectra of the wild type looked like those of high irradiance response and showed peaks at 375, 450, 625 and 725 nm, whereas the action spectra of hy2 showed only the peaks at 375 and 450 nm. Monochromatic light of wavelengths longer than 500 nm had no significant inhibitory effects on hy2 plants. Blue and UV-A light were about five times more effective in the wild type than in hy2 plants. Severe inhibitory effects were observed with UV-B light. It is concluded that inhibition of the growth of the hypocotyl involves combined actions of phytochrome and a putative blue/UV-A photoreceptor(s).     

EXCITATION AND FLUORESCENCE SPECTRA OF THE CHROMOPHORE ASSOCIATED WITH THE DNA-PHOTOREACTIVATING ENZYME FROM THE BLUE–GREEN ALGA ANACYSTIS NIDULANS

DNA-PHOTOREACTIVATING enzymes can be classified as deoxyribonucleate cyclobutane dipyrimidine photolyases*. Such an enzyme was recently purified 3760-fold from the blue-green alga Anacystis niduluns [8]. The absorption spectrum of the enzyme revealed a small peak at 418 nm that was attributed to an impurity. The enzyme has now been purified further, by affinity chromatography on far-ultraviolet (far-u.v.) irradiated DNA non-covalently bonded to cellulose, and its excitation and fluorescence spectra measured. These spectra reveal the presence of a non protein chromophore associated with the algal photolyase. The peak wavelengths in the excitation and absorption spectra in the visible region are almost identical and close to that observed in the in vitro photoreactivation action spectrum [8], observations supporting the view that this chromophore is involved as a cofactor in DNA photo reactivation.     

RELATIVE CONTRIBUTIONS OF TRYPTOPHAN and TYROSINE TO THE PHOSPHORESCENCE EMISSION OF HUMAN SERUM ALBUMIN AT LOW TEMPERATURES

Abstract— Phosphorescence emission and excitation spectra, as well as decay profiles of human serum albumin, were investigated in the wavelength regions of the tryptophan and tyrosine absorption and emission spectra in potassium phosphate buffer at 77 K. Emission and excitation spectra were found to be linear superpositions of the contributions of the tryptophan and tyrosine residues. It is suggested, therefore, that there is no significant tyrosine to tryptophan energy transfer in this protein at low temperature. The phosphorescence decay is, in general, multiexponential with lifetime components of 5.95, 2.7, and 1.2 s. The longest lifetime is characteristic of tryptophan, whereas the two short components are attributed to two types of tyrosine residues located in different environments within the protein. The latter is confirmed by a detailed analysis of the phosphorescence decay profiles determined at different emission wavelengths, and utilizing different wavelengths of excitation favoring either the tryptophan or tyrosine residues.