IN VIVO FLUORESCENCE SPECTROSCOPY OF CHLOROPHYLL IN VARIOUS UNRIPE AND RIPE FRUIT

—Low temperature (77 K) fluorescence emission spectra of slices obtained from the peel and various layers of the pericarp were recorded for fruits which remain green or undergo color break during ripening.
Fluorescence emission peaks characteristic of the photosystem II antennae (λ_F 686 nm) and reaction center (λ_F 696 nm), as well as of the photosystem I antenna (λ_F 730-740 nm), were present in the peel and all parts of the green pericarp of ripe kiwi, avocado and cantaloupe, as well as in ripe tomato and tangerine after color break. The pattern of the fluorescence emission spectra of all samples except that of the kiwi fruit was similar to that obtained from green photosynthetic tissue of leaves, indicating a normal organization of the chlorophyll-containing complexes of thylakoidal membranes. This pattern is characterized by a significantly higher emission at 730-740 nm relative to that of the 696 and 686 nm peaks. In contradistinction, the fluorescence emission at 686 and 696 nm was higher than that at 730 nm in the kiwi fruit, indicating a reduction in the size of the photosystem I antenna chlorophyll. In the innermost yellowish layers of the kiwi pericarp, a further loss of this antenna occurred, as well as disorganization of the photosystem II complex. The above conclusions are suggested also by measurements of variable fluorescence kinetics.
The results presented here indicate that fluorescence spectroscopy might be used as a tool for the study of chlorophyll organization during the growth and ripening periods of fruit.     

FLUENCE-RATE DEPENDENCE OF MONOPHOTONIC REACTIONS OF NUCLEIC ACIDS IN VITRO AND IN VIVO

The Bunsen-Roscoe law, also known as the reciprocity law ( E = f(F) with F = I t ) has only limited validity for monophotonic reactions of nucleic acids. Especially at low fluence rates, the extent of in vitro and in vivo photoreactions of nucleic acids in the far-UV and near-UV range is a function of the fluence and of the fluence rate ( E = f (F;I)). In vitro experiments with poly(dA)poly(dT) clearly show that the far-UV (254 nm) response, indicated by the changes of the ellipticity at 315 nm, does not obey the Bunsen-Roscoe law at low fluence rates in the range between 1 W m^-2 and 20 W m^-2. In vivo experiments with Escherichia coli revealed very similar anomalies. Studying the growth delay after irradiation with far-UV light at 280 nm or near-UV light at 334 nm, we have confirmed the lack of reciprocity in both spectral ranges. The failure of the Bunsen-Roscoe law for the 280 nm and 334 nm UV irradiation effect at low fluence rates was in the range O < I < 40 W m^-2. In both cases reciprocity occurred at higher fluence rates (40 < I < 100 W m^-2).     

AN INSTRUMENT FOR ACTION SPECTRUM STUDIES IN DERMATOLOGY

Abstract— An instrument designed for convenient determination of action spectra for cutaneous photo-responses in man and experimental animals is described. Light from 450 W Xe lamp is dispersed by a concave holographic grating. The spectrum from 244 to 616 nm is projected as a planar strip (2 times 17 cm) intercepted by a grid with 31 ports. The bandwidth at each port is 12 nm and the size of the port increases from about 4 × 4 mm to 6 × 8 mm from the low to high wavelength limits, respectively. Typical fluence rates in quanta m^-2 s^-1 are 4.0 times 10¹⁹ at 298 nm, 16 times 10¹⁹ at 394nm and 22 times 10¹⁹ at 538 nm. Responses due to delayed erythema in normal skin and to musk ambrette photoallergy and solar urticaria in patients skin have been elicited with this instrument.     

ACTION SPECTRA FOR PHOTOTROPIC BALANCE IN Phycomyces blakesleeanus: DEPENDENCE ON REFERENCE WAVELENGTH and INTENSITY RANGE

Abstract— Action spectra for phototropic balance of Phycomyces blakesleeanus sporangiophores were measured for various reference wavelengths and intensity ranges. Balance action spectra were made at fluence rates of 10^-4 W m^-2 with reference wavelengths of 450 nm, 394 nm, 507 nm, and broadband blue light. For broad-blue light and 450 nm light as references, typical flavin-like action spectra were found with a ma jor peak at 455 nm, a secondary peak at 477 nm, and a minor peak at 383 nm; these peaks are wider for broad blue than for 450 nm light. With the 394 nm reference, there is a major peak at 455 nm, a secondary peak at 477 nm and a minor peak at 394 nm. An action spectrum with 507 nm reference has a major peak at 455 nm and a minor peak at 383 nm, but no peak at 477 nm. A balance action spectrum was made with 450 nm reference light near threshold intensity (2 times 10^-8 W m^-2); there, the 386 nm peak is greatly reduced, while the 455 nm peak is enhanced. The intensity dependence of the 386 nm peak was studied in detail for reference light of 450 nm. We found that the relative quantum efficiency of the 386 nm light increases with the logarithm of the 450 nm fluence rate; in the high intensity range (0.3 W m^-2) the relative quantum efficiency of the 386 nm light is 1.3 and approaches zero at 10^-9 W m^-2. These findings indicate that P. blakesleeanus phototropism is mediated by multiple interacting pigments or by a photochromic photoreceptor.     

CHEMILUMINESCENCE IN THE PHOTOOXIDATION OF HUMIC ACIDS*

Abstract— Photo-irradiation of aqueous basic solutions of soil humic acids and synthetic melanins with UV and visible light (Λ > 320 nm) under oxygen or nitrogen atmospheres generates electronic exicted states and radicals. These processes give rise to a long-lived chemiluminescence with emission maxima at 480–500, 570 and 615–650 nm, as well as a paramagnetic resonance (EPR) signal with g-value at 2.006 and ΔH～ 3Gs. Chemiluminescence intensity and EPR signals follow multistep kinetics. An increase of the ratio of OD at 260/400 nm and 400/600 nm and a decrease of amplitude of an EPR signal after prolonged photo-irradiation were observed. Long irradiation also causes a decrease of fluorescence intensity bands with maxima at 535 nm and 495 nm (Λ_ex 480 and 400 nm, respectively), and an increase of the short wavelength band with a maximum at 450 nm (Λ_ex 260 nm). The data indicate that a complex chain of reactions initiated by reactive species leads to the degradation of the aromatic core of the polymers. Oxygen efficiently enhances the chemiluminescence intensity and the rate of photodegradation. The mechanism of photodegradative oxidation and chemiluminescence probably involves an energy transfer process and singlet oxygen formation. The possibility of its occurrence in nature and its significance are discussed.     

PHOTOBLEACHING OF A CYANINE DYE IN SOLUTION AND IN MEMBRANES

Abstract— N,N'-bis(2-ethyl-1,3-dioxolane)-kryptocyanine (EDKC), a lipophilic dye with a delocalized positive charge, photosensitizes cells to visible irradiation. In phosphate-buffered saline (PBS), EDKC absorbs maximally at 700 nm (ε= 1.2 × 10⁵ M⁻¹ cm⁻¹) and in methanol, the absorption maximum is at 706 nm (ε= 2.3 × 10⁵ M⁻¹ cm⁻¹). EDKC partitions from PBS into small unilamellar liposomes prepared from saturated phospholipids and into membranes prepared from red blood cells (RBC) and binds to human serum albumin (HSA). The EDKC fluorescence maximum red shifts from 713 nm in PBS to 720–725 nm in liposomes and RBC membranes and the fluorescence intensity is enhanced by factors of 14–35 compared to PBS (φ= 0.0046). EDKC is thermally unstable in PBS (T_1/2= 2 h at 1.3 × 10⁻⁵ M EDKC), but stable in methanol. In liposomes and RBC membranes, EDKC is 10 times more stable than in PBS, indicating that it is only partially exposed to the aqueous phase. Quenching of EDKC fluorescence in liposomes and RBC membranes by trinitrobenzene sulfonate also indicates that EDKC is not buried within the membranes. Photodecomposition of EDKC was oxygen-dependent and occurred with a low quantum yield (6.4 × 10⁻⁴ in PBS). Singlet oxygen was not detected upon irradiation of EDKC in membranes or with HSA since the self-sensitized oxidation of EDKC occurred at the same rate in D₂O as in H₂O and was not quenched by sodium azide or histidine.     

INDUCTION OF SKIN TUMORS IN THE RAT BY SINGLE EXPOSURE TO ULTRAVIOLET RADIATION

Abstract— The dose response for tumor induction in albino rat skin by single exposures of UV radiation has been characterized. The shaved dorsal skin of 202 animals was exposed to either of two sources: one emitting a broad spectrum of wavelengths from 275 to 375 nm, and the other emitting at 254 nm. Skin tumors began to appear within 10 weeks of exposure and continued to appear for 70 weeks. The highest tumor yield was 5.5 tumors per rat and occurred when the rats were exposed to 13.0 times 10⁴ J/m² of the 275–375 nm UV. The 275–375 nm UV was about eight times as effective as the 254 nm UV for the induction of tumors throughout the exposure range from 0.8 times 10⁴ to 26.0 times 10⁴J/m². Tissue destruction and hair follicle damage was found at the highest exposure to 275–375 nm UV but at none of the exposures to 254 nm UV. Repeated weekly exposures to 275–375 nm UV proved less effective than an equivalent single exposure for inducing tumors, even though the multiple exposures caused more severe skin damage. The transmission of the UV through excised samples of rat epidermis indicated that the exposure to the basal cell layer was about 3% of the surface exposure at 254 nm and about 15% of the surface exposure between 275 and 320 nm. The dependence of tumor yield on UV exposure was linear for 254 nm UV but was more complex for the 275–375 nm UV. For the latter more tumors were produced per unit exposure at lower exposures than at higher exposures.     

THE ROLE OF 4-THIOURIDINE IN LETHAL EFFECTS AND IN DNA BACKBONE BREAKAGE CAUSED BY 334 nm ULTRAVIOLET LIGHT IN Escherichia coli

Strains of Escherichia coli that lack 4-thiouridine (⁴Srd) are killed by monochromatic 334 nm UV light (UV) less efficiently than their wild-type parents, which contain ⁴Srd. Oxygen enhancement ratios (OER) at 10% survival are 3.3 for a strain that possesses ⁴Srd, and 2.6 for one that lacks ⁴Srd. Single-strand breaks in DNA caused by 334 nm UV accumulate more than twice as fast in the wild-type strains than in the strains lacking ⁴Srd. The results suggest that ⁴Srd is an important chromophore in some near-UV lethal effects. The results also suggest that the excitation energy from 334 nm UV light may be passed from RNA to DNA, resulting in single-strand breaks.     

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.     

OPTICAL PROPERTIES OF PHYTOCHROME IN THE BLUE- SPECTRAL REGION AS MEASURED IN VIVO AND IN VITRO

In the blue spectral region, the phototransformation difference spectrum of oat phytochrome extracted as P_fr differs from that of phytochrome extracted as P_r. The difference absorbance maximum for phytochrome extracted as P_fr is at 420 nm, while that extracted as P_r is at 412 nm. The phototransformation difference spectrum measured in the blue in oat coleoptile tips without inner leaves, corresponds very well with that of phytochrome as extracted in its P_fr form. There is, however, a slight apparent attenuation of the blue difference band relative to those in the red-far-red. In coleoptile tissue containing inner leaves, the blue difference band is relatively even more highly attenuated. A similar attenuation is observed in the blue, in the protochlorophyllide to chlorophyllide phototransformation difference spectrum. In the spectrum measured with excised coleoptile without inner leaves, there is a small attenuation, while in coleptile tissue with inner leaves the attentuation is nearly 9-fold. These data suggest that the observed attenuation is probably artifactual. Neither instrumental non-linearity nor fluorescence induced by the measuring beam could explain the observed attenuation. It is suggested that the observed attenuation is probably mainly the result of wavelength dependent scatter amplification, the amplification in the blue being attenuated by the high background absorption of other pigments in this region.     

ACTION SPECTRUM FOR ERYTHEMA IN HUMANS INVESTIGATED WITH DYE LASERS

Abstract— Erythema reactions of human skin were reevaluated with improved experimental methods: a tunable, highly monochromatic irradiation source as well as an instrumental measurement of skin reactions were used. The irradiation system consisted of an excimer laser pumped dye laser and a U V fiber optic system. The skin color after irradiation was determined with a colorimeter in the three-dimensional norm system of the Commission Internationale d'Eclairage (CIE). The wavelength dependence for delayed erythema was investigated in the UVB and UVA region from 294 nm to 374 nm in skin type II and III individuals. The maximum of the action spectrum in the UVB range was measured at 298.5 nm and an additional maximum was found at 362 nm in the UVA range. The action spectrum is compared with previous spectra from the literature and with the current standard erythema curve of the CIE as well as with other photobiological action spectra. Our results suggest a UVA/UVB boundary at 330 nm.     

UVA PHOTOLYSIS USING THE PROTEIN-BOUND SENSITIZERS PRESENT IN HUMAN LENS

Abstract —This research was undertaken to demonstrate that the protein-bound chromophores in aged human lens can act as sensitizers for protein damage by UVA light. The water-insoluble (WI) proteins from pooled human and bovine lenses were solubilized by sonication in water and illuminated with UV light similar in output to that transmitted by the cornea. Analysis of the irradiated proteins showed a linear decrease in sulfiydryl groups with a 30% loss after 2 h. No loss was seen when native a-crystallin was irradiated under the same conditions. A 25% loss of histidine residues was also observed with the human lens WI fraction, and sodium dodecyl sulfate polyacrylamide gels indicated considerable protein cross-linking. Similar photodamage was seen with a WI fraction from old bovine lenses. While the data show the presence of UVA sensitizers, some histidine destruction and protein cross-linking were also obtained with a-crystallin and with lysozyme, which argue that part of the histidine loss in the human WISS was likely due to tryptophan acting as a sensitizer.
A preparation of human WI proteins was irradiated with a total of 200 J/cm² of absorbed light at 10 nm intervals from 290 to 400 nm. Photodamage of cysteine SH groups (35%) and methionine (28Y0) was maximum at 330 nm and diminished linearly at longer wavelengths. The major loss of tryptophan (80%) occurred at 290 nm, but destruction was observed throughout the UVA range. Tyrosine was 35% destroyed at 290 nm but decreased sharply to only 50 at 330 nm. A constant loss of histidine (20%) was seen at all wavelengths from 290 to 360 nm, with some loss (7–8%) even at 400 nm. These action spectra show that the human lens WI fraction contains a collection of protein-bound UVA sensitizers that can cause protein photodamage similar to that seen in cataractous lenses.     

TIME RESOLUTION OF A BACK PHOTOREACTION IN BACTERIORHODOPSIN

Abstract— The back photoreaction from the M(412nm) intermediate in the photocycle of light-adapted bacteriorhodopsin, BR_LA(570 nm), is studied using pulsed laser excitation. The decay of a primarily produced species, M_P, regenerates BR_LA(570nm) in a process characterized by a half life of 200 ns at 25°C. The absorption maximum of M_P is blue shifted (Λ_max≃ 395 nm) relative to that of M(412nm). The primary photochemical step, M(412nm) → M_P, is attributed to a conformational change in the polyene residue. The energy and entropy of activation of the subsequent M_P→ BR_LA (570 nm) relaxation are reported and discussed.     

LIGHT INDUCED FLUORESCENCE SPECTRAL CHANGES IN NATIVE PHYTOCHROME FROM Secale cereale L. AT LIQUID NITROGEN TEMPERATURE

Abstract— Fluorescence spectra of native rye phytochrome were determined under different light conditions at liquid nitrogen temperature. Fluorescence spectrum of the red-light-absorbing form (Pr) had a major peak at about 685 nm (14 600 cm⁻¹) and a broad sub-peak at about 515 nm (19 400 cm⁻¹). The peak height at 685 nm was reduced by irradiation with monochromatic light of 640 nm, and a new peak became obvious at about 702 nm (14250 cm⁻¹). This spectral change was almost completely reversed by subsequent irradiation with 700-nm light. Fluorescence spectrum of the photoequilibrium mixture of Pr and far-red-light absorbing form under continuous red light showed a sharp peak at about 685 nm having a peak height ca. 12% of Pr, and a broad sub-peak at about 508 nm (19 700 cm⁻¹). Light of 730 nm did not reduce the peak height at about 685 nm but induced a new shoulder at about 699 nm (14300 cm⁻¹). Monochromatic light of 640 and 700 nm given following the light of 730 nm could not reverse the spectral change at 699 nm induced by the irradiation with 730-nm light. Fluorescence spectrum of Pr in partially degraded phytochrome was similar to that in native phytochrome but the peak position in the red region was shifted by about 5 nm (100 cm⁻¹) to the blue.     

AN EXPERIMENTAL STUDY OF THE CHANGES IN PIGMENTATION IN HUMAN SKIN IN VIVO WITH VISIBLE AND NEAR INFRARED LIGHT

Abstract— The skin of the lower inner arm of volunteers was irradiated, with a 390–1700 nm light source, through a fiber optic bundle for times of up to 1.2 × 10⁴ s and with powers of up to 0.35 W/cm². Simultaneously with the irradiation, spectra (390–720 nm) of the remitted intensity were measured, while a 5.0 cm in diameter area of the skin around the fiber bundle was maintained at constant temperature, within 0.2°C. The generation of a photoproduct was observed and measured as changes in the remitted intensity within 600 s (10 min) of the start of irradiation.
The photoproduct formed was characterized by a weak absorption in the blue part of the spectrum (400–450 nm), leading to a bluish appearance in the irradiated area only. The color change appears as a two step process. It starts with a "soluble" photoproduct, which disappears, within 24 h after irradiation, and an "insoluble" photoproduct which appears with irradiation greater than 3 ×10³ s (50 min). No spectral differences were detected between the two photoproducts. The "insoluble" photoproduct persists for periods of up to 8 weeks. The color change in the skin is immediate and there is no erythema associated with this color change.     

SYNERGISTIC LETHAL ACTION OF ULTRAVIOLET-VIOLET RADIATIONS AND MILD HEAT IN ESCHERICHIA COLI

Abstract— The lethal interaction of far ultraviolet (254nm), near ultraviolet (334 and 365nm) and violet visible (405nm) radiation treatment with mild heat treatment was studied. Except at 254nm, a strong positive radiation dose-dependent interaction (synergism) was always observed. The efficiency of sensitisation to heat, as a function of dose at each wavelength, was found to be directly correlated with the dose necessary to eliminate the shoulder from the survival curve of a repair proficient strain but was apparently unrelated to the relative near-ultraviolet sensitivities of a repair deficient strain. The interaction was independent of the order of treatments. A radiation dose of 10⁶ Jm^-2 at 365nm slightly sensitised a cell population to 45°C incubation (normally non-lethal) and strongly sensitised the cells to 48°C treatment (normally 80 percent survival after 2 hours). It is proposed that in addition to DNA damage, both heat treatment and near ultraviolet treatment interfere with DNA recovery mechanisms so that the combination of the two agents inevitably leads to a strong positive interaction.     

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.     

ENERGY TRANSFER FROM CHLOROPHYLL b TO CHLOROPHYLL a IN A HYDROPHOBIC MODEL SYSTEM

Abstract— Chlorophyll a and chlorophyll b purified by high-performance liquid chromatography (HPLC) were subsequently adsorbed on the surface of a pellicular reverse phase packing normally used in HPLC. The granule surface is reacted with octadecyl groups and furnishes an hydrophobic substrate for pigment adsorption. Reflectance spectra of chlorophyll a and chlorophyll b , each adsorbed at average spacings of about 11 nm² per molecule, had red region maxima at 664 and 643nm respectively. Fluorescence excitation spectra for 740nm emission from these surfaces peaked at about 420nm for chlorophyll a and 460nm for chlorophyll b. Adsorbed pigments excited at either of the two wave lengths had a single fluorescence emission peak at 683nm for chlorophyll a and at 664nm for chlorophyll b. A surface having both pigments adsorbed in approximately equal amounts with an overall average spacing of about 5.6nm² per molecule also had peaks at 420 and 460nm in the excitation spectrum. However, excitation of adsorbed molecules on this (latter) surface, at either 420 or 460nm, produced emission with the single chlorophyll a peak at 683nm. It is concluded that, under the conditions of our experiment, exciting adsorbed chlorophyll b contributes strongly to emission from adsorbed chlorophyll a.     

HYDRATED ELECTRON FORMATION IN NANOSECOND and PICOSECOND LASER FLASH PHOTOLYSIS OF HEMATOPORPHYRIN IN AQUEOUS SOLUTION

Nanosecond (lambda exc = 266, 355 and 532 nm) and picosecond (lambda exc = 355 nm) laser flash photolysis of hematoporphyrin (Hp) was performed in neutral (pH 7.4) and alkaline (pH 12) aqueous solution, as well as in the presence of 0.1% Triton X-100. The dependence of the yield of photoproduced hydrated electrons (e-aq) on laser pulse energy was studied over a wide range of energies (0.2 to greater than 1000 mJ cm-2). The results show that e-aq are predominantly formed in a two-photon process at lambda exc = 266 and 355 nm. One-photon quantum yields are higher at lambda exc = 266 nm than at lambda exc = 355 nm. Both one-photon and two-photon pathways are less efficient at higher Hp concentration, reflecting the influence of Hp self-aggregation. Two-photon e-aq formation is more efficient when 30 ps pulses are used for excitation, as compared to 10 ns pulses. No e-aq could be detected at lambda exc = 532 nm. Nanosecond pulse-induced transient spectra obtained at pH 7.4 are also discussed.     

STEPS IN THE POPULATION OF THE EMITTER IN THE BACTERIAL LUCIFERASE REACTION

Abstract— The reaction of luciferase-bound flavin hydroperoxide with both I-¹H and 1–²H decanal has been examined at 2°C in both low (0.01 M ) and high (0.35 M ) phosphate buffer, pH 7, where the kinetics and deuterium isotope effects are quite different. Upon reaction in both buffers there are rapid (<2 ms) increases in absorption at both 380 and W nm, followed by decay over the subsequent seconds and minutes. The changes at 380 nm exhibit a primary isotope effect and are rapid compared to bioluminescence, indicating that the scission of the aldehyde C — H bond occurs prior to the step responsible for populating the electronically excited state. However, the final absorbance change at 600 nm decays in parallel to bioluminescence under the several different conditions studied, suggesting the involvement of a long-wavelength absorbing flavin species in the production of, the excited state. Evidence is also presented indicating that under certain conditions there may be two (sequential) steps, each of which exhibits a primary isotope effect involving the same H atom.