RESOLUTION OF SPECTRAL AND LIFETIME HETEROGENEITY OF TRYPTOPHAN FLUORESCENCE IN BACTERIORHODOPSIN

Abstract— The picosecond time-resolved fluorescence decay of bacteriorhodopsin (BR) was analyzed by the maximum entropy method. Results showed five distributions of lifetimes indicating at least five decay components. A wavelength-dependent study of emission decay of BR was carried out in the wavelength region from 310 to 390 nm. The decay at each wavelength was resolvable into four decay components by the discrete exponential analysis. The three short lifetime components (100 ± 20 ps, 400 ± 50 ps and 1.0 ± 0.1 ns) were independent of wavelength, whereas the longest lifetime component was wavelength dependent (varying from 4.1 ns at 310 nm to 5.7 ns at 390 nm). These results are inconsistent with the existing model of associating the fluorescence of bacteriorhodopsin with two or four lifetime components. An attempt is made to associate the five decay components with the emitting tryptophans of BR.     

NANOSECOND LASER PHOTOLYSIS OF RHODOPSIN AND ISORHODOPSIN

Kinetic and spectral measurements have been carried out on the primary intermediate in the photolysis of rhodopsin and isorhodopsin, initiated by a 457 nm, 6 ns (FWHM) laser pulse. In rhodopsin the kinetic decay of bathorhodopsin was found to be 140 ± 15 ns at 20°C. The decay of bathorhodopsin to lumirhodopsin has an activation energy of 51 ± 4 kJ/mol (12.2 ± 1 kcal/mol). The decay kinetics of bathorhodopsin were found to be the same for rhodopsin in membrane and detergent solubilized suspensions. The kinetic decay of the batho product in the photolysis of isorhodopsin was found to be the same as rhodopsin.
The corrected transient spectrum 50 ns following excitation in rhodopsin has two peaks near 560 and 440 nm. A peak was also observed in isorhodopsin near 550 nm at 50 ns following excitation but no transient was observed in the blue. The 550 nm peak in isorhodopsin has an intensity similar to that in rhodopsin indicating that the quantum yields for the formation of batho products of rhodopsin and isorhodopsin are similar under the irradiation conditions used here. Transient spectra for rhodopsin and isorhodopsin 1 μs following excitation are also different. In isorhodopsin the corrected transient spectrum has a peak at 500 nm, similar to low temperature steady state irradiation spectra. The 1 μs transient spectrum in rhodopsin is more intense than in isorhodopsin and shows a peak at 475 nm.     

聚对苯二甲酸乙二酯荧光光谱的激发波长依赖性研究

聚对苯二甲酸乙二酯（PET）的荧光光谱已经得到了相当广泛和深入的研究,当激发光波长位于310～360nm时,可以观察到位于360～390nm范围的荧光发光,这部分荧光的来源有很多争议,至今未取得一致的看法,有作者认为,此处的荧光发光是PET链段上苯环之间的相互作用形成的基态二聚体（ground-state dimer）引起的发光;还有人认为这是苯环基团之间相互作用形成的激基缔合物的发光（excimeric emission）。     

WAVELENGTH DEPENDENCE FOR THE INACTIVATION OF ATP IN THE VACUUM-ULTRAVIOLET REGION ABOVE 140 nm

Radiation effects were investigated on the activity and the structure of adenosine triphosphate in the wavelength range from 140 nm to 260 nm, using monochromatized synchrotron radiation from the INS-SOR storage ring. The sample was irradiated as a thin film in vacuum. The activity of adenosine triphosphate decreased sharply below 180 nm as judged by the luminescence in the luciferin-luciferase assay. From the exponential decay of function, the cross-section for inactivation was calculated to be of the order of 10^-21 m²/photon in the range from 140 to 170 nm. No decrease was detected at wavelengths of 190 nm and above. The calculated quantum yield increased as the wavelength became shorter and reached to 0.20 at 150 nm. The release of adenine at 160 nm-irradiation was detected by thin layer chromatography; no adenosine diphosphate or adenosine monophosphate occurred. Only a trace of adenine was found after 190 nm-irradiation. These results indicate that the broad absorption peak for higher excitations attributable to the base moiety around 190 nm does not cause both structural and functional changes, while the absorption by the sugar-phosphate group produces the rupture of N -glycosidic bond, and probably leads to the loss of function.     

FLUORESCENCE OF TRYPTOPHAN IN KERATIN

The excitation and emission spectra have been determined for the fluorescence from trypto-phan residues in dry keratin. The fluorescence decay was also measured and shown to be a single exponential with a rather long lifetime of 6.9 ns. It is suggested that the emission takes place from a state formed by interaction between the ¹L_a state of the tryptophan residues and neighbouring polar or polarizable groups in the protein. The fluorescence excitation spectrum displays a peak at 290 nm, and its appearance at this position rather than at 280 nm, which is the absorption maximum of tryptophan, is believed to arise from inner filtering by the tyrosine residues in keratin.     

PICOSECOND FLUORESCENCE KINETICS IN SPINACH CHLOROPLASTS AT LOW TEMPERATURE

Abstract— At 77 K the fluorescence from spinach chloroplasts excited using picosecond mode-locked laser pulses at 620 nm is made up of 5 separate kinetic components. Three of these are predominant at short wavelengths. between 650 and 690 nm, and they appear to correspond to the 3 decay phases seen at room temperature. The 2 new components. a 100 ps rise and a 3000 ps decay, characterize the longer (730–770 nm) wavelength fluorescence. The temperature dependence of the kinetic components of the long wavelength fluorescence shows that the 3000 ps decay accounts for essentially all of the large increase in fluorescence yield observed at low temperature. Furthermore, it appears that this increase does not result entirely from an increase in the fluorescence lifetime, as has been proposed. The dependences of these 2 new components (the 100 ps rise and 3000 ps decay) on emission wavelength and temperature are similar enough to suggest they have a common origin, presumably the chlorophyll pigment component C705. The amplitudes (yield/lifetime) of these 2 phases are approximately equal, and they are opposite in sign. Thus. we see evidence of time-resolved excitation transfer from those pigment molecules that absorb the 620 nm radiation to those that give rise to the long wavelength fluorescence at low temperature.     

KINETIC STUDIES OF THE TWO LIGHT REACTIONS IN PHOTOSYNTHESIS

Abstract— The decay kinetics of the photo-induced absorbance changes in red and green algae are very sensitive to the wavelength of the actinic light. A four to tenfold increase in half-decay time is noted in going from short wavelength (550–650 mμ) to long wavelength (> 700 mμ) excitation. The slow decay rates produced by long wavelength light can be enhanced with a steady background of short wavelength light. A relationship between initial decay rates and O₂ evolution rates is described. This relationship allows a direct correspondence between these spectroscopic studies and the 'red-drop' and 'enhancement' experiments of Emerson.     

Effect of CdS Interlayer on Properties of CdTe Based Quantum Dots

Highly luminescent thioglycolic acid-capped CdTe-based core/shell quantum dots (QDs) were synthesized through encapsulating CdTe QDs in various inorganic shells including CdS, ZnS and CdZnS. CdTe/CdS core/shell QDs exhibited a significant redshift of emission peaks (a maximum emission peak of 652 nm for the core/shell QDs and 575 nm for CdTe cores) with increasing shell thickness. In contrast, the redshift of photoluminescence (PL) peak wavelength of CdTe/ZnS QDs was less than 15 nm. The PL peak wavelengths of the core/shell QDs depended strongly on core size and shell thickness. The PL quantum yields (QYs) of the CdTe/CdS core/shell QDs are up to 67 % while that of CdTe/ZnS core/shell QDs is 45 %. A composite CdZnS shell made CdTe cores a high PL QY up to 51 % and broadly adjusted PL spectra (a maximum PL peak wavelength of 664 nm). The epitaxial growth of the shell was confirmed by X-ray powder diffraction analysis and luminescence decay experiments. Because of high PL QYs, tunable PL spectra, and low toxicity from a ZnS surface layer, CdTe/CdZnS core/shell QDs will be great potential for bioapplications.     

胞嘧啶水溶液体系辐解的瞬态产物研究

Using the ns pulse radolysis, we studied the characteristic absorption spectrum and kinetic decay of cytosine anion radical (Cyt-). Results showed that the characteristic absorption of Cyt- was located at λ=355±5 nm, and decayed following the first order kinetics with τ1/2=265 ns at pH=7.0. The decay became slower and τ1/2 rapidly rised with the increment of pH value, Cyt- protonated at C6 in acidic solution, and the characteristic absorption was located at λ=310±5 nm, and decayed following the second order kinetics: Cyt- protonated at N3 in aqueous solution of pH≥7, and the characteristic absorption was located at λ=295±5 nm, and decayed following the second order kinetics.     

Fluorescence reabsorption in anthracene single crystals: Lifetime variations with emission wavelength and temperature

Fluorescence spectra and lifetimes of anthracene melt-grown single crystals and sublimation flakes have been examined at 298 and 77°K, using a mono-photon counting technique for the lifetime measurements. The observed emission decay times were nearly independent of the excitation wavelength, though a small dependence of the fluorescence spectrum on the excitation wavelength was noted. By contrast, large variations of fluorescence lifetimes in thick crystals were found as a function of emission wavelength. For thick melt-grown single crystals at 298°K the lifetime was found to increase from 9.8 nsec at 405 nm to 20.4 nsec at 445 nm. For sublimation flakes at 77°K and at 298°K and for thick melt-grown crystals at 77°K, the lifetimes were less than 10 nsec and were nearly independent of emission wavelength. Despite these relatively large variations in lifetimes, the decay rates at each separate wavelength remained exponential, within experimental error. Theoretical calculations were made of emission lifetimes based on a model with one reabsorbing state. The calculations are in substantial agreement with the experimental results.     

FLUORESCENCE OF HOUSEFLY VISUAL PIGMENT

Abstract —The fluorescence of housefly photoreceptors was studied in vivo by using the deep pseudopupil technique. Whereas the rhodopsin R490 of the peripheral retinular cells fluoresces negligibly the metarhodopsin M580 fluoresces distinctly in the red. The newly discovered metarhodopsin M'is produced by intense blue light and can be reconverted into rhodopsin by intense long wavelength light. M'also fluoresces in the red; its excitation spectrum and emission spectrum peak at _max= 570 and 660 nm respectively.
Intense ultraviolet light irreversibly reduces the visual pigment fluorescence as well as the broad band autofluorescence (kmnx 470 nm) originating from non-visual pigments in the fly's eye.     

Shock wave induced decomposition of RDX: time-resolved spectroscopy

Time-resolved optical spectroscopy was used to examine chemical decomposition of RDX crystals shocked along the [111] orientation to peak stresses between 7 and 20 GPa. Shock-induced emission, produced by decomposition intermediates, was observed over a broad spectral range from 350 to 850 nm. A threshold in the emission response of RDX was found at about 10 GPa peak stress. Below this threshold, the emission spectrum remained unchanged during shock compression. Above 10 GPa, the emission spectrum changed with a long wavelength component dominating the spectrum. The long wavelength emission is attributed to the formation of NO2 radicals. Above the 10 GPa threshold, the spectrally integrated intensity increased significantly, suggesting the acceleration of chemical decomposition. This acceleration is attributed to bimolecular reactions between unreacted RDX and free radicals. These results provide a significant experimental foundation for further development of a decomposition mechanism for shocked RDX (following paper in this issue).     

PHYTOCHROME INTERMEDIATES IN VIVO-I. EFFECTS OF TEMPERATURE, LIGHT INTENSITY, WAVELENGTH AND OXYGEN ON INTERMEDIATE ACCUMULATION

Abstract— Accumulation of weakly absorbing phytochrome intermediates has been demonstrated in Pisum epicotyl tissue under conditions of pigment cycling using a quasi-continuous measuring spectrophotometer. An action spectrum shows 690–700 nm to be the most efficient wavelength range in this process. Difference spectra for the decay of intermediates maintained by 690 nm light show that, if the experiment is done at 0°C, only P_fr is formed. At – 11°C, intermediates decaying to P_r can also be observed. At – 20°C, P_r is produced as well as a pigment with peak absorption at 710nm. Kinetic analysis of intermediate decay at – 11°C reveals that at least two intermediates are maintained by 690 nm light. The level of intermediate maintained by incandescent light at 0°C was 25% higher in air than in nitrogen.     

DESIGN AND PERFORMANCE OF THE OKAZAKI LARGE SPECTROGRAPH FOR PHOTOBIOLOGICAL RESEARCH

A computer-operated spectrograph was recently built at Okazaki, Japan. Different specimens can be placed on a horseshoe-shaped focal curve (10 m long) covering a wavelength range of 250 to 1000 nm so they can be irradiated simultaneously. The linear dispersion is about 0.8 nm/cm. The photon fluence rate on the focal curve is 5 x 10¹⁵. photons x cm^-2 x s^-1 at 300nm and 1 x 10¹⁶ photons x cm^-2 x s^-1 at 600 and at 900 nm. The spectral half width is 5.5 nm or less on the focal curve. The stray light content is about 10^-5 of the main peak at the peak wavelength ± 100 nm. Specimens are set in microcomputer-controlled threshold boxes so that wavelengths, photon fluence rates, photon fluences and timing of irradiations are controlled automatically according to a pre-programmed schedule. An optical fiber system is also provided for remote irradiations.     

Cholesteric mixture containing a chiral azobenzene-based dopant: material with reversible photoswitching of the pitch of the helix

A new low molar mass chiral-photochromic dopant was synthesized. It contains a menthyl fragment as the chiral group and an azobenzene group, capable of E - Z photoisomerization, as the photochromic component. The substance obtained was used as a chiral dopant in mixtures with a comb-shaped cholesteric acrylic copolymer with menthyl-containing chiral side groups and phenyl benzoate nematogenic side groups. Such mixtures form a cholesteric mesophase. The chiral dopant led to an additional twisting of the cholesteric helix, i.e. to a shift of the selective light reflection peak to a shorter wavelength region of the spectrum. The initial copolymer gave selective light reflection in the spectral range 1200-1400 nm; the mixture containing 3.5 mol % of chiral-photochromic dopant reflects light with λ_max~ 850 nm. The action of light with λ_ir~ 440 nm results in E - Z isomerization of the azo-group of the chiral dopant and in a shift of the selective light reflection peak to the long wavelength region of the spectrum (amplitude of shift = 30 nm). This is explained by a lower helical twisting power of the Z-isomer of the chiral dopant. This process is thermally reversible: annealing of irradiated films leads to a back shift of the selective light reflection peak to the short wavelength region of the spectrum due to Z - E isomerization. Kinetic features of the direct and backward processes of isomerization were studied: it was shown, that mixtures of the chiralphotochromic azobenzene-containing dopant with cholesteric polymers give new possibilities for the creation of polymer materials with a reversibly regulated helical supramolecular structure which determines their optical properties.     

ACTION SPECTRUM FOR INACTIVATION OF THE INFECTIVITY OF POTATO VIRUS X BY U.V. RADIATION

Abstract— Quantum yields for inactivation of infectivity of potato virus X by monochromatic ultraviolet radiation of wavelengths ranging from 230 to 290 nm, were measured with reference to energy absorbed by (a) the whole virus and (b) the virus RNA. The yields depended on the wavelength, but those with reference to energy absorbed by the RNA varied much less (with extreme values of 10^-3 and 1.9 ± 10^-3 than those with reference to whole virus. Consequently the action spectrum for inactivation of a dilute solution of the virus resembled the shape of the absorption spectrum of the RNA, but not closely enough to allow coincidence by adjusting the scales. The amount of photoreactivation increased as the wavelength increased and also as the year progressed from May to July; the extreme values of the photoreactivable sector were 0.43 and 0.86.     

EVIDENCE FOR THE FORMATION OF A CHLOROPHYLL a/ZEAXANTHIN COMPLEX IN LECITHIN LIPOSOMES FROM FLUORESCENCE DECAY KINETICS

The interaction of Chi a with zeaxanthin (Zea), which is an analogue of lutein, has been studied in soya bean lecithin liposomes using the fluorescence of Chi as monitor. The fluorescence emission spectrum at 4.2 K of Chi a showed characteristic changes in the presence of Zea: the emission maximum shifted from 688 nm to 680 nm, and a peak at 731 nm appeared. The fluorescence decay kinetics of Chi a alone could be described by the sum of two exponential components (T₁,≅0.8 ns, T₂≅2.5 ns). In the presence of Zea a component with a long lifetime, T≅5 ns, appeared with a large relative amplitude (40%). This indicated the formation of a Chl a /Zea complex, in which Chl a /Chl a interaction is negligible, presumably because of strong interaction between Chl a and Zea. The fluorescence anisotropy decay kinetics supported the hypothesis of the formation of a large Chl a containing complex in the presence of Zea. A rotational correlation time, φ≅14 ns at 4°C and φ≅21 ns at 30°C, was found, which is distinctly larger than for samples containing Chl a only. We interpret these results as further evidence for a strong interaction between Chl a and Zea in the hydrophobic environment of the lecithin liposomes. This interaction may also occur in the Chl-proteins of the Chi alb light-harvesting complex of plant photosynthesis.     

Experimental Correspondence between Spore Dosimetry and Spectral Photometry of Solar Ultraviolet Radiation

Abstract— The biologically effective dose of solar UV radiation was estimated from the inactivation of UV-sensitive Bacillus subtilis spores. Two types of independent measurements were carried out concurrently at the Aerological Observatory in Tsukuba: one was the direct measurement of colony-forming survival that provided the inactivation dose per minute (ID/min) and the other was the measurement of the spectral irradiance by a Brewer spectrophotometer. To obtain the effective spectrum, the irradiance for each 1 nm wavelength interval from 290 to 400 nm was multiplied with the efficiency for inactivation derived from the inactivation action spectrum of identically prepared spore samples. Integration of the effective spectrum provided the estimate for ID/min. The observed values of ID/min were closely concordant with the calculated values for the data obtained in four afternoons in 1993. The average ratio (±SD) between them was 1.24 (±0.16) for 14 data points showing high inactivation rates (<0.05 ID/min). Considering difficulties in the absolute dosimetry of UV radiation, the concordance was satisfactory and improved credibility of the two types of monitoring systems of biologically effective dose of solar UV radiation.     

LONG WAVELENGTH UV RADIATION AFFECTS CHEMILUMINESCENCE OF HUMAN POLYMORPHONUCLEAR LEUCOCYTES

Abstract— Luminol-enhanced chemiluminescence in suspensions of human polymorphonuclear leucocytes stimulated with the chemotactic oligopeptide formyl-methionyl-leucyl-phenylalanine was increased by exposure of the cells to long wavelength UV radiation (mainly320–400 nm). Leucocytes treated with 0.3 × 10⁴ J m^-2 of UV responded with doubled peak values, and 4 × 10⁴ J m^-2 lead to a five-fold increase in peak chemiluminescence, as compared with non-exposed cells. Supernatants isolated from irradiated leucocytes contained increased amounts of both myeloperoxidase and lactate dehydrogenase and showed higher chemiluminescence values, when compared with supernatants from sham-irradiated cells. The results suggest that leucocyte degranulation contributes to the inflammatory properties of long wavelength UV.     

PICOSECOND LASER PHOTOLYSIS OF SQUID RHODOPSIN AT ROOM AND LOW TEMPERATURES

Abstract. Squid rhodopsin extracted with 2% digitonin (pH 10.5 or 7.0) was excited with a 347 nm light pulse from a mode-locked ruby laser at room temperature. Within 19 ps after the excitation, absorbance at 430 nm due to hypsorhodopsin increased and subsequently decreased with a decay time of 45 ± 10 ps. Absorbance at 550 nm due to bathorhodopsin increased with a rise time of 50 ± 10 ps. These results are the first observations of hypsorhodopsin at room temperature and clearly show that hypsorhodopsin is a precursor of bathorhodopsin which has been considered to be the earliest photoproduct in the photobleaching process of rhodopsin.
Hypsorhodopsin appeared with a rise time of 70 ± 10 ps at 421 nm at liquid nitrogen temperature without any bathorhodopsin being observed during the formation of hypsorhodopsin. An experiment using an N₂ laser showed that squid bathorhodopsin converted to lumirhodopsin with a decay time of about 300 ns at room temperature.