IN VIVO MEASUREMENTS OF THE PHYTOCHROME PHOTOSTATIONARY STATE IN FAR RED LIGHT

Abstract— The in vivo photostationary state, φ_fr= ([ P _fr]_fr/[ P ]), of phytochrome in far red light has been determined in mustard seedling cotyledons by three different methods. The φ_fr is a function of the length of time of etiolation ( t = 36 hr, φ_fr= 0·14; t = 72 – 120 hr, φ_fr= 0·075). The calculated φ_r= 0·8. The amount of P _tot is strongly dependent on the time of onset of far red light. These data imply that it would be almost impossible to maintain a constant level of P _fr in mustard cotyledons over a considerable period of time.     

COMPARATIVE PHOTOCHEMICAL ANALYSIS OF HIGHLY PURIFIED 124 KILODALTON OAT and RYE PHYTOCHROMES in vitro

Abstract A direct comparison of the photochemical interconversions between red (P_r-) and far-red (P_fr-) absorbing forms of highly-purified 124 kDa oat and rye phytochromes under identical experimental conditions was performed. In two different buffer systems at 5°C, the quantum yields for the P_r to P_tr and P_fr to P_r phototransformations under constant red and far-red illumination, φ _r and φ_fr respectively, were determined to be 0.152-0.154 and 0.060-0.065 for oat preparations and 0.172-0.174 and 0.074-0.078 for rye preparations. These values as well as the wavelength dependence of the photoequilibrium produced under continuous illumination throughout the visible and near-ultraviolet spectrum were based on the absorption spectra of the two phytochrome preparations and revised molar absorption coefficients. The molar absorption coefficients were estimated by quantitative amino acid analysis and shown to be identical for the two monocot phytochromes (i.e. 132 mM ⁻¹ cm⁻¹ at the red absorption maximum for the P_r form). Because these measurements were performed under identical experimental conditions, including buffer, temperature, light fluence rate, and instrumentation, the differences observed must reflect structural features inherent to the two different monocotyledonous phytochromes.     

THE TEMPERATURE DEPENDENCE OF PHYTOCHROME TRANSFORMATIONS

Abstract— The kinetics of phytochrome transformation were examined over a 30°C temperature range (+5° to–25°C) in 75% glycerol. Two new intermediate reaction stages are described for the transformation of the red-absorbing form, P _r to the far-red absorbing form, P _fr The free energies, enthalpies and entropies of activation were obtained for five of the six reaction stages observed in the transformation of P _r to P _fr and for the two reaction stages observed in the reverse process. All exhibited positive entropies of activation with the maximum being 25 entropy units. The results suggest that the phototransformations between P _r and P _fr consist of relaxation processes beginning with the intermediate produced immediately upon absorption of a quantum of light and ending with either P _r or P _fr.     

Fluorescent Dimers of Merocyanine 540 (MC540) in the Gel Phase of Phosphatidylcholine Liposomes

Abstract— Fluorescence emission from merocyanine 540 (MC540) dimers was observed in dipalmitoylphosphatidylcholine (DPPC) vesicles. This unusual behavior was observed only for vesicles in the gel-phase state. No dimer fluorescence was observed either in monopalmitoylphosphati-dylcholine (C₁₆PC) micelles or in liquid-crystalline DPPC vesicles, indicating that dimer fluorescence efficiency increases in highly packed interfaces. The excitonic theory of Kasha was used to interpret the spectral features. The overall fluorescence quantum yield (φ_r) decreases with decreasing lipid: probe ratio, not only because of the presence of a weakly fluorescent dimer that absorbs a high fraction of the total absorbed light but also due to quenching of monomer emission. This suggests the existence of probe domains. The dimer fluorescence quantum yields (φ_m) were estimated in DPPC large unilamellar vesicles (LUV) and DPPC multilamellar vesicles. The dependence of φ_r with probe concentration is compatible with values of φ_m lower than 0.05. The dimerization equilibrium of MC540 in C₁₆PC micelles and DPPC-LUV was also studied. Apparent dimerization equilibrium constants, K_dapp and dimer absorption spectrum were calculated in C₁₆PC micelles for the first time. The dimerization equilibrium constant in DPPC-LUV was calculated and discussed in terms of the fraction of volume occupied by the lipid phase.     

RED LIGHT INTERACTIONS WITH BLUE AND ULTRAVIOLET LIGHT IN POLAROTROPISM OF GERMLINGS OF A FERN AND A LIVERWORT

Abstract— In polarotropism of the chloronema of the fern Dryopteris filix-mas (L.) Schott and of the germ tube of the liverwort Sphaerocarpos donnellii Aust. a phytochrome action in blue and u.v. was presumed[1, 2]. In the present paper this assumption was tested by simultaneously irradiating with red and blue, and red and near u.v. Red energy is given to shift the phytochrome photoequilibrium in favour of high P _fr/ P ^total concentrations. The data obtained by simultaneous irradiation are consistent with the predictions made under the assumption of a phytochrome involvement in the blue- and u.v.-mediated polarotropic response.     

Pfr PHYTOCHROME AND SUCROSE REQUIREMENT FOR RHYTHMIC LEAFLET MOVEMENT IN ALBIZZIA

Abstract— Albizzia leaflets open and close with a circadian periodicity without damping during extended dark periods if supplied with sucrose and irradiated with red each rhythmic cycle. In the absence of a carbohydrate source, the rhythm damps irrespective of phytochrome status and the differential effects of red and far-red light treatments tend to disappear after 2 or 3 cycles. The rhythm also damps if leaflets are exposed to red only once, or if they are exposed to far-red once or cyclically. Sucrose does not prevent such damping but affects the stable angle assumed; the rhythm damps in the closed position in the presence of sucrose, and in the partially open position in its absence. Sucrose appears to have two distinct effects; it interacts with P _fr to promote manifestation of the rhythm and it reduces the average angle, whether the P _fr level is low or high.     

STIMULATION OF THE SHIBATA SHIFT BY PHOTOCHROME IN THE COTYLEDONS OF THE MUSTARD SEEDLING SINAPIS ALBA L.

Abstract— In the cotyledons of the mustard seedling Sinapis alba L. the duration of the Shibata shift can be greatly shortened by a pretreatment with light pulses prior to the protochlorophyllide– chloro-phyllide a photoconversion. It was shown that the light pulses act through photochrome (P _fr ). Since reversibility of a red light pulse induction by a far-red light pulse is rapidly lost (within 2 min) it is concluded that at least the initial action of P_fr occurs rapidly in this response. On the other hand, the effect of a red light pulse on the rate of protochlorophyll regeneration in the mustard seedling cotyledons is fully reversible by a far-red light pulse for more than 5 min. It is concluded that control of protochlorophyll regeneration and control of the Shibata shift by phytochrome cannot be consequences of the same initial action of P_fr Apparently P_fr controls both phenomena independently.     

A LASER FLASH PHOTOLYSIS STUDY OF PYRENE-1-ALDEHYDE. INTERSYSTEM CROSSING EFFICIENCY, PHOTOREACTIVITY AND TRIPLET STATE PROPERTIES IN VARIOUS SOLVENTS

Abstract— Triplet-and singlet-related photoprocesses of pyrene-1-aldehyde (PA) in various solvents have been investigated in detail using 337.1 and 355 nm laser flash photolysis in conjunction with time-correlated determination of fluorescence lifetimes (τ_F) and steady-state photochemical and absorption-emission spectral measurements. In benzene, the lowest triplet of PA (43 < E_T < 46 kcal/mol) has a lifetime of about 50 µs (τ_T) and displays the absorption maximum at 443 nm with a maximum extinction coefficient (ε_max) of 21000 M ^-1cm^-1; the corresponding ketyl radical has a sharp absorption maximum at 428 nm (ε_max≥ 25000 M ^-1cm^-1). The quantum yields (φ_T) of lowest triplet occupation are high in nonprotic solvents (0.6–0.8), decrease in protic solvents (alcohols) as the polarity of the latter is increased, and maintain a complementary relationship with the quantum yields (φ_F) of fluorescence. Quantum yields (φ_PC) of loss of PA due to photoreactions in some solvents have also been determined under conditions of steady irradiation at 366 nm; φ_PC is in the range 0.1–0.2 in electron-rich olefinic solvents such as cyclohexene and tetramethylethylene. These results concerning τ_F, τ_T, φ_F. φ_T and φ_PC as well as the effects of 1,2,4-trimethoxybenzene and 2,5-dimethyl-2,4-hexadiene as quenchers for fluorescence, triplet yield, and photochemistry are discussed in the light of possible state orders for PA in polar and nonpolar environments.     

THE INFLUENCE OF MOLECULAR CONFORMATION ON THE STABILITY OF ULTRAVIOLET STABILIZERS TOWARD DIRECT AND DYE-SENSITIZED PHOTOIRRADIATION: THE CASE OF 2-(2'-HYDROXY-5'-METHYPHENYL)BENZOTRIAZOLE (TIN P)

Abstract— The mechanism for photodegradation of the ultraviolet photostabilizer 2-(2'-hydroxy-5'-methylphenyl)benzotriazole (TIN P) upon direct and dye-sensitized (singlet molecular oxygen [O₂(1Δ_g)]-mediated) irradiation was studied. From the experimental TIN P photodegradation rate data, and low temperature (77 K) fluorescence and phosphorescence quantum yields, one can conclude that the photodegradative process involves phosphorescent states of TIN P. The open conformer of TIN P quenches O₂(¹Δ_g) by physical scavenging with a rate constant (k_q) in dimethylsulfoxide of 2.8 times 10⁶ M ^-1 s^-1. The intramolecular hydrogen-bonded conformer does not appreciably interact with O₂(¹Δ_g). In the presence of a relatively high concentration of OH- (either 5 times 10^-2 M KOH in ethanol or water at pH 13), the ionic form of TIN P (with an ionized phenol group) physically and chemically quenches O₂(¹Δ_g). The reaction rate constant ( k _r) is 1 times 10 ⁸ M ^-1 s^-1, and the ratio k _q/ k _r is approximately three in alkaline aqueous media.     

MECHANISM OF THE PHOTOCHEMICAL REACTION OF DIPHENYLAMINES WITH CARBON TETRACHLORIDE

Abstract. The quantum yields of HCI (φ_HC1) formation have been measured for the photolysis of N -methyldiphenylamine (MeDPA), triphenylamine (TPA) and diphenylamine (DPA) in the presence of CCl₄ in polar solvents. The quantum yields of N-methylcarbazole formation (φ_mφca) have also been determined for the system MeDPA-CCl₄. With increasing CCl₄ concentration, φ_HCl increases as φ_MeCA decreases, and φ_HCl reaches maximum values 2.7 at 1 M CCl₄. Using laser photolysis, transient spectra have been recorded for MeDPA in the absence and presence of CCl₄ in polar and non-polar solvents, and for TPA. Transient absorption due to the triplet states and photocyclization products (without CCU), exciplexes, the (C₆H₅)₂ NCHi radical, the MeDPA⁺ cation radical, the (TPA⁺., CCl₄) ion pair, and the TPA⁺ cation radical have been identified. The mechanistic implications of these results are discussed.     

Irradiation of DNA with 193 nm Light Yields Formamidopyrimidine-DNA Glycosylase (Fpg) Protein-Sensitive Lesions

Abstract— Irradiation of aqueous solutions of plasmid DNA (pUC18) at pH 7.6 with 193 nm laser light results in low yields of prompt single strand breakage (air-saturated sample φ_ssb= [1.5 ± 0.1] ± 10⁻⁴, argon-saturated sample φ_ssb= [0.9 ± 0.1] ± 10⁻⁴). Treatment of the irradiated DNA samples with Escherichia coli formamidopyrimi-dine-DNA glycosylase (Fpg) protein results in an approximate 20-fold increase in the yield of single strand breakage (air-saturated sample φ_fpg= [33.1 ± 3.1] ± 10⁻⁴, argon-saturated sample φ_fpg= [23.8 ± 2.6] × 10 ⁴). This result indicates that 193 nm light induces other modification) (most likely of the purine moieties) that are 20 times more abundant than prompt strand breakage within the DNA matrix.     

POTENTIAL PHOTOSENSITIZERS FOR PHOTODYNAMIC TUMOR THERAPY—I. PHOTOPHYSICAL PROPERTIES OF TWO CHLORIN DERIVATIVES

Abstract— Photophysical properties of two chlorin type molecules (CHLI) and (CHLII) were investigated in different solvents. Quantum yields of fluorescence φ_F of S, → T, intersystem crossing φ_T, and of singlet oxygen (¹Δ_g) formation φ_Δ, as well as the Stern-Volmer constants for the quenching of the S, states by oxygen and the bimolecular rate constants of quenching of ¹Δ_g by the chlorins were measured. The values of φ_T and φ_Λ can be given as 0.57 and 0.58 for CHLI and 0.69 and 0.58 for CHLII. The values of the fluorescence quantum yields, the strong absorption of the chlorins in the red (Λ > 630 nm) and the high values of the quantum yields for ¹Δ_g formation recommend the chlorin derivatives as potential markers and photosensitizers for tumor therapy.     

PHOTOLYSIS OF AQUEOUS SOLUTIONS OF POTASSIUM CIS-DIAQUOBIS (OXALATO) CHROMATE (III)

Abstract— The photolysis of aqueous solutions of cis -[Cr(C₂O₄)₂(H₂O)₂]- at 254 nm and pH 4 produced CO₂ and H₂ in nearly equal yields. The quantum yield of hydrogen, φ², increased by 9% and the yield of carbon dioxide, φ, by 65% as the pH was lowered from 4 to I. The total gas yield, φ_gas, decreased in the presence of added oxalate or chromium (II) ions and when the light intensity was lowered. The gas yield in D₂O was appreciably higher than in H₂O. The variation of φ_gas with pH (D) and with added oxalate ion was roughly parallel in the two liquid media. The gas yield increased in the series:
A tentative mechanism is suggested to explain the results.     

QUANTIFICATION OF THE SOLVENT EFFECTS ON THE TRIPLET QUANTUM YIELD OF PSORALEN BY THE "LINEAR SOLVATION ENERGY RELATIONSHIP"

Triplet formation quantum yields (Φ_τ) of psoralen in a set of 17 pure solvents ranging from n -hexane to water and in dioxane: water mixtures were obtained by nanosecond laser flash photolysis. The triplet yield increases with solvent polarity. The extremum values are 0.009 and 0.545 in n -hexane and water, respectively. Good correlations of the experimental Φ_τ values with empirical "polarity" scales (Dimroth/Reichardt's E_T [30], Kamlet/Taft's solva-tochromic parameters β, and α, and Swains acity/basity A_S/B_S) were obtained: Ln(φ_T^-1 - 1) = 8.86 - 0.143E_T(30) Ln(φ_T^-1 - 1) = 4.40 - 2.34_τ - 1.70α Ln(φ_T^-1 - 1) = 4.65 - 3.72A_s - 1.12B_s The results are discussed in terms of the sensitivity of psoralen triplet quantum yield to solvent polarity and hydrogen-bonding abilities.     

PHOTOCONTROL OF PHYTOCHROME DESTRUCTION AND BINDING IN DICOTYLEDONOUS vs MONOCOTYLE-DONOUS SEEDLINGS. THE INFLUENCE OF WAVELENGTH AND IRRADIANCE

Abstract— The irradiance and wavelength dependence of phytochrome destruction in vivo was analysed in etiolated cotyledons of Cucurbita pepo L. and etiolated seedlings of Amaranthus caudatus L. In contrast to grass seedlings, the rate of P _tot destruction could only be saturated by light sources that establish relatively high P _fr levels (about 50% of total phytochrome, corresponding to the photostationary state established by 693 nm light). To explain the irradiance dependence of P _tot destruction in dicots at irradiances above 0.1 Wm^-2, where the light reaction is at least one order of magnitude faster than P _fr destruction, we suggest there is a fast intercalary dark reaction between photoreaction and destruction. This dark reaction is probably—as in grass seedlings—the binding of P _fr to a receptor site. We conclude that the differences between dicots and grass seedlings with respect to the phytochrome system are of a quantitative rather than a qualitative nature.     

ACTINOMETRY IN MONOCHROMATIC FLASH PHOTOLYSIS: THE EXTINCTION COEFFICIENT OF TRIPLET BENZOPHENONE AND QUANTUM YIELD OF TRIPLET ZINC TETRAPHENYL PORPHYRIN

Abstract— The extinction coefficient ε_T, of triplet benzophenone in benzene has been directly determined by absolute measurements of absorbed energy and triplet absorbance, Δ D ⁰_T, under demonstrably linear conditions where incident excitation energy, E ₀, and ground state absorbance, A ₀, are both extrapolated to zero. The result, 7220 ± 320 M ^-1 cm^-1 at 530 nm, validates and slightly corrects many measurements relative to benzophenone of triplet extinction coefficients made by the energy transfer technique, and of triplet yields obtained by the comparative method.
As E ₀ and A ₀ both decrease, Δ D ⁰_T becomes proportional to their product. In this situation, the ratio R = (1/ A ₀)(dΔ D ⁰_T/d E ₀) = (ε_T - ε_G)φ_T. Measurements of R , referred to benzophenone, give (ε_T - ε_G)φ_T for any substance, without necessity for absolute energy calibration.
Both absolute and relative laser flash measurements on zinc tetraphenyl porphyrin (ε_T - ε_G at 470 nm = 7.3 × 10⁴ M ^-1 cm^-1) give φ_T= 0.83 ± 0.04.     

THE PHOTOCHEMISTRY OF RIBOFLAVIN—VI. THE PHOTOPHYSICAL PROPERTIES OF ISOALLOXAZINES

Abstract— Many of the photophysical properties of riboflavin and several other N-10 substituted isoal-loxazines have been measured and these include: quantum yields of fluorescence at 77 K and 298 K, φ, quantum yields of phosphorescence at 77 K, φ_P, lifetimes of the triplet state by electron spin resonance and phosphorescence at 77 K, φp,. and the quantum yield of intersystem crossing. φ_isc. For riboflavin in an alcoholic matrix at 77 K the limiting values were: φ _J = 0.32, φ_P= 0.007, φp = 0.20s and φ_isc= 0.7. At 298 K, φ _f for riboflavin in water and alcohol were 0.25 and 0.32, respectively. The results for the photophysical processes are compared with several photochemical processes known to involve the triplet state of riboflavin in aqueous solution. It is concluded that the φ _isc decreases from 0.7 in alcohol to 0.6 in water at 298 K.     

PHOTOCHEMISTRY OF FLAVINS. II. PHOTOPHYSICAL PROPERTIES OF ALLOXAZINES AND ISOALLOXAZINES

Abstract. Pulsed laser photolysis at 347nm has been used to study the transient spectroscopy of alloxazine, lumichrome, lumiflavin, and riboflavin in acidic (pH 2.2) aqueous solution and in ethanol. Intersystem crossing quantum yields (φ_ISC) were determined by a modification of the comparative laser excitation method which utilizes the variation of the triplet yield with intensity in conjunction with a kinetic model for the various photophysical and photochemical processes occurring during the pulse. Fluorescence quantum yields and lifetimes are also reported. Correction for quenching of the excited singlet state by H⁺ ions shows that, in neutral aqueous solution, intersystem crossing for flavins is an efficient process (φ_ISC˜ 0.7) which, in conjunction with fluorescence, accounts for the fate of all absorbed photons. For alloxazine (φ_ISC˜ 0.45) and lumichrome (φ_ISC˜ 0.7) the results are more difficult to interpret owing to interconversion between alloxazine and isoalloxazine structures in the singlet excited state. For all four compounds, the quantum yield of products derived from the singlet excited state is estimated as ˜0.04. There is evidence of biphotonic product formation at high laser energies. In ethanol, where φ_ISC for lumichrome is about twice that of lumiflavin, internal conversion between the excited singlet and ground states appears to be a significant process. Complete triplet-triplet absorption spectra in the region 260–750nm are reported. For lumichrome at pH 2.2 there is spectral evidence for isomeric triplet states which appear to be in equilibrium.     

1H-INDENYLFURAN AND THIOPHENE DERIVATIVES– A NEW CLASS OF SINGLET OXYGEN SENSITIZERS

Abstract— Photophysical and photochemical properties of 1H-indenylfuran and thiophene derivatives were studied. UV spectra showed that all the compounds tested absorbed UV light in the region 350–380 nm. The fluorescence spectra of 4a-e showed bands in the region 410–470 nm and quantum yields (φ_F) in the range 0.25–0.88. Attempts to calculate the triplet energy of 4a-e failed except for 4c and 4d , which showed E_T= 43–44 kcal mol^-1. These compounds are a new class of singlet oxygen sensitizers. The sensitized reaction of 2,5-dimethylfuran with singlet oxygen was followed showing that 4a and 4e are more reactive than α-terthiophene (4.0 and 2.5 times respectively), while the other compounds show the same reactivity as 1 . This behavior can be explained considering the different intersystem crossing quantum yields of compounds 4a-e . Diazabicyclo[2. 2. 2]octane is a quencher of singlet oxygen in this reaction, while we can exclude superoxide ion formation using the photooxidation of α, α-dimethylstilbene.     

POTENTIAL PHOTOSENSITIZERS FOR PHOTODYNAMIC THERAPY—II. PHOTOPHYSICAL PROPERTIES OF [26] PORPHYRIN

Abstract— –Photophysical properties of [26] porphyrin (26 P) were investigated in chloroform. The quantum yields of fluorescence, of S₁→ T₁ intersystem crossing and singlet oxygen formation were measured. The purity, stability, the strong absorption in the red (δ_max= 783 nm; ε_max= 28 000 M ¹ cm^-1) and the ability of singlet oxygen formation recommend 26 P as potential photosensitizer for tumor therapy.