PHYTOCHROME INTERMEDIATES IN VIVO–II. CHARACTERISATION OF INTERMEDIATES BY DIFFERENCE SPECTROPHOTOMETRY

Abstract— In epicotyl tissue of Pisum , irradiation of P_r at – 196°C forms a stable product P₆₉₈, whereas P_fr forms a stable product P₆₅₀. On warming P₆₉₈, dark transformation to P_r predominates. On warming P₆₅₀ to – 70°C an intermediate P₆₉₀ is formed which bleaches on further warming to –10°C. When tissue is cooled to –196°C during actinic irradiation, difference spectra for subsequent warming to –10°C indicate that P_r, P_fr and an intermediate P₇₁₀ are formed from weakly absorbing intermediates. Complete photoconversion of P_r to P_fr is not possible at temperatures below –5°C. As the temperature is reduced, the amount of P_fr produced from P_r decreases, while P₇₁₀ increases. P₇₁₀ can be photoconverted at –20°C and above, ultimately forming P_r, but in contrast to P_fr it is not photoconvertible at –196°C.     

SOLVENT EFFECTS ON THE TAUTOMERIZATION OF 5'-DEOXYPYRIDOXAL. A PHOTOPHYSICAL STUDY

Abstract— Absorption and fluorescence spectra of 5'-deoxypyridoxal (DPL) in various pure solvents and mixtures were recorded both at room temperature and over the range10–65°C. The areas under the absorption bands were analyzed to obtain the mole fraction (f_N, f_z) of two tautomers (the zwitterionic, Z, and neutral, N, forms) in the ground state. The following spectral parameters were determined from the fluorescence spectra: Stokes shift (Δ v ), fluorescence quantum yield of the neutral form (Q_N), fluorescence ratio of the neutral to the zwitterionic form (ø_N/ø_Z) and the rate constant of tautomerization ( k ₁) from Z to N in the excited state. Some of these parameters (f_N, Δ v , Q_N, k ₁) were found to depend on the proton donor character of the solvent, whereas others (ø_N/ø_Z) depended on its dipole moment. Thus, the absorption and fluorescence spectra of DPL allow one to obtain information on the polarity and the concentration of –OH groups on its environment.     

DOES MEMBRANE ATTACKING PHOTODYNAMIC ACTION REFLECT THE SO-CALLED PHASE TRANSITION?

Abstract— With Saccharomyces cerevisiae cells the temperature dependence of toluidine blue (TB) sensitized photodynamic inactivation was measured in the range 10–33°C using either H₂O (pH 7.9) or 45% D₂O (pH 6.8) as a solvent. Plots of ln(1/LD₅₀) (LD₅₀: light dose for 50% survival) as a function of the reciprocal of temperature could be fitted by two straight lines with a discontinuity at 21–22°C. In view of previous findings on TB-cell interaction that TB sensitizes the cell, after long incubation, in the membrane rather than from outside the cell, it is suggested that the present results reflect the phase transition of lipid matrix.     

MULTIPLE ACTION OF FAR-RED LIGHT IN PHOTOPERIODIC INDUCTION and CIRCADIAN RHYTHMICITY

Abstract— It is generally accepted that phytochrome influences the photoperiodic induction of flowering through its interaction with the circadian clock mechanism. We have attempted to separate the effects of phytochrome on the clock mechanism from those that mediate flowering directly by examining a number of responses that are unrelated to flowering but are also regulated by the circadian clock. Gas exchange measurements of both CO₂ and H₂0 vapor were monitored under light conditions (200 μmol m ² s⁻¹) where the addition of far-red energy is required for the maximal promotion of flowering. In addition, photosynthetic capacity and maximal transpiration rates were measured in plants grown under continuous dim (20 μmol m⁻² S⁻') light, with or without supplemental far-red, by exposing them briefly to saturating fluxes (1000 μmol m⁻² s^-l) of light. Net CO₂ fixation was very weakly rhythmic in plants grown under both high and low light and this weak oscillation was completely suppressed by far-red light. Far-red also suppressed the rhythm in transpiration under high light, but the rhythm was immediately reinstated when the far-red light was removed. The phase of this rhythm was also reset with the next peak always occurring15–18 h after the far-red was turned off. When grown under dim light, the transpiration rhythm was not suppressed and the amplitude of the oscillation was more than doubled. Far-red light appears to interact with the rhythm in transpiration in a manner suggesting that the stomatal rhythm may be coupled to the same clock oscillator that regulates the flowering rhythm.     

CIRCADIAN RHYTHM OF Robinia pseudoacacia LEAFLET MOVEMENTS: ROLE OF CALCIUM AND PHYTOCHROME

Abstract— The effect of external calcium level, calcium ionophore A23187 and red light on the circadian rhythm of Robinia pseudoacacia leaflet movements has been studied. Fifteen minute red light pulses shifted the phase of leaflet rhythmic movement with a phase-response curve type 0. Maximum advances and delays (about 10 h and 8 h, respectively) were obtained between circadian time (CT) 10 and CT 12 at the end of a subjective day. An almost null effect was obtained at the end of a subjective night. Phytochrome is the photoreceptor involved in phase shifting since this effect of red light is reversed by 5 min of far red light. Two hour pulses of external calcium, applied as CaCl₂ (10 m M ), and 2 h pulses of calcium ionophore A23187 (10–50 μM) also shifted the phase of leaflet circadian movement and caused the same type of phase-response curve, with maximum advances and delays at the same time as those produced by red light. Two hour pulses of an external calcium chelator, EGTA (5 m M ), and a calcium channel blocker, LaCl₃ (10–50 m M ), damped the circadian rhythm or did not change the phase when they were applied at lower concentration. These results indicate that phytochrome could control the circadian oscillator, which drives Robinia leaflet movements by increasing the intracellular calcium concentration.     

LIGHT-INDUCED QUENCHING OF PHOTOSYSTEM II FLUORESCENCE AT 77 K

Abstract— Light-induced quenching of the low temperature fluorescence emission from photosystem II (PS II) at 695 nm ( F ₆₉₅) has been observed in chloroplasts and whole leaves of spinach. Photosystem I (PS I) fluorescence emission at 735 nm ( F ₇₃₅) is quenched to a lesser degree but this quenching is thought to originate from PS II and is manifest in a reduced amount of excitation energy available for spillover to PS I. Differential quenching of these two fluorescence emissions leads to an increase in the F ₇₃₅/ F ₆₈₅ ratio on exposure to light at 77 K. Rewarming the sample from -196°C discharges the thermoluminescence Z-band and much of the original unquenched fluorescence is recovered. The relationship between the thermoluminescence Z-band and the quenching of the low temperature fluorescence emission ( F ₆₉₅) is discussed with respect to the formation of reduced pheophytin in the PS II reaction center at 77 K.     

PHOTODICHROISM OF RHODOPSIN SOLUTIONS AT -196°C

Abstract— The photodichroism of a system of randomly oriented, bleachable pigment molecules, rigidly fixed in an inert transparent matrix was investigated theoretically. A formula was derived for the photochemical equilibrium reaction between three pigments, N ₁⇆ N ₂⇆ N ₃ in the case that the absorption ellipsoids of the pigments are rotationally symmetric and their symmetry axes coincide. The formula was applied to the dichroism induced by plane polarized light of different wavelengths in an aqueous rhodopsin-glycerol mixture at –196°C. It was found that the absorption ellipsoids of this visual pigment, its analogue, isorhodopsin, and its first bleaching product, prelumirhodopsin, are elongated with an apparent axial ratio of about 5. It was concluded that the light absorbing properties of these pigments cannot be described by a single transition moment vector (linear absorber). The absolute value of the quantum efficiency of the conversion of rhodopsin to prelumirhodopsin was shown to be approximately equal to the quantum efficiency of bleaching rhodopsin at room temperature. Some evidence was obtained that indicates that the relative quantum efficiencies of the rhodopsin system at – 196°C may be wavelength dependent.     

EFFECT OF TEMPERATURE AND pH ON THE QUENCHING OF TRIPLET LUMIFLAVIN IN THE PRESENCE AND ABSENCE OF FERRI- AND FERROCYANIDE

Abstract— –Flash photolysis at 450 nm over the temperature range 0.8–60°C was used to determine Arrhenius parameters for the first and second order disappearance of triplet lumiflavin (1.66 µ .M ) at a flash energy of 2 kj in deaerated phosphate buffer at varying pH:
³Lf → Lf₀
³Lf +³Lf → Lf₀+ Lf₀
Arrhenius parameters were also determined for the pseudo first-order quenching of triplet lumiflavin by 10 µ M ferri- and ferrocyanide ions,
³Lf + Fe³⁺→ Fe³⁺→ Lf₀+ Fe³⁺ (energy transfer)
³Lf + Fe²⁺→ Lf^-+ Fe³⁺ (electron transfer)
and for disappearance of the semireduced lumiflavin in the presence of ferrocyanide at pH 6.8, by the second-order reaction
Lf^-+Lf ^-→ Lf₀+ Lf⁼.     

SOLID PHASE PHOTOREDUCTION OF METHYLVIOLOGEN ON CELLULOSE SENSITIZED BY TRIS(2,2'-BIPYRIDYL)RUTHENIUM COMPLEX

–Methylviologen (MV²⁺) adsorbed on cellulose could be reduced photochemically in the solid phase sensitized by tris(2,2'-bipyridyl)ruthenium(II) complex, [Ru(II)(bpy)₃], using disodium ethylene-diaminetetraacetic acid (Na₂EDTA) as a reducing agent. Formation of the cation radical (MV ⁺.) was confirmed by visible and EPR spectra. The MV⁺. formed on cellulose was remarkably stable against air oxidation and rapidly accumulated even by the irradiation under air. Water adsorbed on the cellulose greatly retarded the photoreaction. Action spectrum showed that the excitation of Ru(II)(byp)₃ is responsible for the photochemical reaction. The quenching of the emission from Ru(II)(bpy)*₃ by MV²⁺. indicated that a primary photochemical reaction occurs between Ru(II)(bpy)*₃ and MV²⁺. The main reaction path is the reduction of MV²⁺ by Ru(II)(bpy)₃, giving MV⁺. and Ru(III)(bpy)₃, followed by the reduction of Ru(III)(bpy)₃ to Ru(II)(bpy)₃ with Na₂EDTA, which in turn is oxidized to give carbon dioxide.     

QUANTITATIVE SPECTROFLUORIMETRY-THE FLUORESCENCE QUANTUM YIELD OF QUININE SULFATE

Abstract— By idealizing the design of a typical spectrofluorimeter that measures fluorescence from dilute solutions, we are able to derive an equation that predicts the dependence of the observable amplitude on the concentration of the solute and on several instrumental parameters. The result of the derivation, the fluorescence function, is used together with measurements of rhodamine B fluorescence and colloidal silica scattering to calibrate a spectrofluorimeter.
The fluorescence function and the calibration data are used to determine the absolute fluorescence quantum yield of quinine sulfate in 0–1 N H₂ So₄. The results, Ø₂₅₀=0.582 with excitation at 250 mμ, adn Ø₃₅₀=0.577 with excitation at 350mμ. agree well with the previous evaluation of Melhuish, Ø₃₄₅=0.546.     

PHOTOCHEMISTRY OF RETINOCHROME

Abstract— Retinochrome is a photopigment found in the visual cells of cephalopods. It has been considered to act as a supplier of the 11- cis -retinal required for synthesis of rhodopsin, because its all-trans chromophore is isomerized to 11- cis form in the light. Light and thermal reactions of squid retinochrome were investigated by low-temperature spectrophotometry.
On irradiation with green light at liquid-nitrogen temperature, retinochrome (λ_max 496 nm, – 190°C) is converted mainly to an intermediate lumiretinochrome (λ_max 475 nm, – 190°C), its chromophore being changed to 11- cis -retinal. On irradiation with blue light at - 190°C, retinochrome is changed to a photosteady–state mixture (λ_max 487 nm, – 190°C) composed mainly of retinochrome and lumiretinochrome, since lumiretinochrome is partially regenerated back to retinochrome. Similarly, irradiation of lumiretinochrome with blue light also results in the same photosteady-state mixture, which can be completely reverted to lumiretinochrome on re-irradiation with green light.
Lumiretinochrome is stable at a wide range of temperatures from – 190°C to about – 20°C. Above – 20°C, it is further converted, thermally, into metaretinochrome (λ_max 470 nm), which is the same bleached product as has been observed on irradiation of retinochrome at room temperatures. Thus, the light-bleaching process of retinochrome is rather simple compared with that of rhodopsin.     

PHYTOCHROME INTERMEDIATES IN VIVO– III KINETIC ANALYSIS OF INTERMEDIATE REACTIONS AT LOW TEMPERATURE

Abstract— Kinetic experiments have provided evidence for a series of light and dark reactions of phytochrome intermediates at low temperature in Pisum epicotyl tissue. A photoequilibrium exists between P_r and P₆₉₈, and between P_fr and P₆₅₀. A dark reversion of P₆₉₈P_r and P₆₅₀p_fr at –70°C has been demonstrated. When cooled to 70°C under incandescent light, most of the phytochrome in the tissue is driven into photochemically unreactive intermediates. About 2% of the phytochrome remains as weakly absorbing intermediates that form P_r and P_fr in darkness. A scheme is presented for phytochrome phototransformation in vivo.     

THE EFFECTS OF LIQUID HOLDING ON NEAR-UV(300–400 nm) IRRADIATED ESCHERICHIA COLI STRAINS DIFFERING IN NEAR AND FAR-UV RADIATION SENSITIVITY

Abstract— Stationary phase cells from four Escherichia coli strains differing in near- (nur vs. nur ₊) and far-UV (recAl vs. recA₊) radiation sensitivity were subjected to near-UV radiation (NUV) in 0.85% saline. Although the NUV-irradiated cultures yielded increased colony numbers following 24 h of liquid holding (LH), a fluctuation test for each experiment showed that the observed increases were not due to recovery but were in fact due to cell multiplication. The decline in viability observed after NUV with liquid holding using the fluctuation test was equivalent in strains RT2, 3 and 4 while the decline observed with RT1 was less marked. The discrepancy between LH involving cell densities of 10₈-10₉ and 1–4 cells/m/ can be resolved by assuming that with dense cell suspensions, NUV-induced membrane damage leads to leakage or lysis, supplying sufficient nutrients to allow growth of undamaged, surviving cells.     

INHIBITION OF ETHYL BENZENE OXIDATION BY STABLE IMINOXY RADICALS, STUDIED BY CHEMILUMINESCENCE

Abstract— The inhibition by stable radicals having the structure (X-O)₂NO of the initiated oxidation of ethyl benzene was studied. According to a formal kinetic scheme, verified by both theoretical computer calculations and an experimental chemiluminescence method, the rate constants of the interaction of peroxyradicals with iminoxy radicals were estimated at 60°C to be: 2.4 times 10⁵ litres mol^-1 s^-1 for X = OCH₃ and 3.4 times 10⁵ litres mol^-1 s^-1 for X = OCH₂CH₃.     

MECHANISM OF FORMATION OF FORMALDEHYDE IN THE FLASH PHOTOLYSIS OF METHYL FORMATE

Abstract —From flash photolyses of methyl formate (HCOOCH₃), d -methyl formate (DCOOCH₃), methyl formate- d ₃ (HCOOCD₃) and fully ***deuterated methyl formate (DCOOCD₃) in the vapour phase at room temperature, the relative efficiencies of the formyl and methoxyl radicals in producing formaldehyde have been determined.     

COMPUTER SIMULATION OF EXCITON JUMPING STATISTICS AND ENERGY FLOW IN C-PHYCOCYANIN OF ALGAE Agmenellum quadruplicatum IN THE PRESENCE OF TRAPS

Abstract—Energy migration has been studied in C-phycocyanin (C-PC) rods with traps located in the terminal trimer disc, using the Monte Carlo method and the system of differential equations. It has been found that jump time statistics can be described by the function F = C(t/0>)exp(-t/ < t_o>), where C is the constant, t and < t₀ > are, respectively, the exciton jump time and its averaged value for chromophores of the corresponding spectral types (α 84 , β84 or β155). The values < t₀ > were calculated for the cases of C-PC monomers, trimers and higher associates.
The C-PC model, which consists of three hexamers with traps located in the β84 chromophores of the peripheral trimer, was examined. It was found that the total efficiency of excitation capturing, ø_tr, exceeds 90%, provided "local" quantum yield of energy trapping ø₀ > 10%. The ø₀ value influences both the excitation lifetime (τ) and the mean number of excitation jumps (N_iump) before its conversion. For the ø₀ = 100% and 10%, the corresponding lifetimes and numbers of jumps were calculated to be τ= 75 and 155 ps and N_jump= 105 and 222 jumps, respectively.
The dynamics of excitation redistribution along the C-PC rods and the fluorescence kinetics for various ø₀ values were calculated for C-PC chromophores excited by a +, and the correlation between these processes and ø ₀ , was disclosed. The transient processes of excitation redistribution were shown to proceed within a time period t < 30 ps.     

THE HABER-WEISS CYCLE

Abstract— The Haber-Weiss cycle:
was investigated at low pH by radiolysis of oxygen or nitrogen saturated solutions of hydrogen peroxide. It was found that reaction 2 has a low rate constant: k ₂= 3.0 ± 0.6 M ^-1 s^-1 (pH 2.3, 22°C). The rate determining step of reaction 2 is most probably the transfer of an electron from a π₈* orbital of HO₂ to the empty _u* orbital of H₂O₂. Overlap between these two orbitals is hindered by the filled π₈* orbitals of H₂O₂. Fe(HI)EDTA catalyses reaction 2.     

Effects of Calmodulin Inhibitors and Blue Light on Rhythmic Movement of Robinia pseudoacacia Leaflets

Abstract— The effect of several calmodulin (CAM) antagonists, blue light and an intracellular calcium inhibitor, on the circadian rhythm of Robinia pseudoacacia leaflet movement has been studied. The CAM antagonists, chlorpromazine (CPZ), trifluoperazine (TFP), calmidazolium and N -(6-aminohexyI)-5-chloro-1-naphthalenesulfonamide (W₇) shifted the phase of the circadian rhythmic movement while W₅, an inactive analogue of W₇, had no effect. Two hour pulses of calmidazolium (10–50 μ M ) gave rise to a phase-response curve with maximum advances (up to 9 h) at circadian time (CT) 6 and maximum delays (up to 7 h) at CT 22. No effect was found on transition from subjective day to subjective night and vice versa. The TFP (10–50 μ M ), applied as 2 h pulses during the circadian cycle, shifted the phase of the circadian leaflet movement and also produced maximum advances in the middle of subjective day. Two hour blue light pulses shifted the phase of leaflet rhythmic movement. The phase-response curve obtained showed maximum advances (up to 5 h) in the middle of subjective day and maximum delays on transition from subjective day to subjective night. Two hour pulses of 50 μ M 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate hypochloride (TMB-8), an intracellular calcium inhibitor, caused the same type of phase-response curve, with maximum advances and delays occurring at the same time as those produced by blue light. These results indicate that CAM might be involved in controlling the circadian oscillator that drives Robinia leaflet movement. The relationship between CAM and calcium with red and blue light is discussed.     

THE Py SCALE OF SOLVENT POLARITIES. SOLVENT EFFECTS ON THE VIBRONIC FINE STRUCTURE OF PYRENE FLUORESCENCE and EMPIRICAL CORRELATIONS WITH ET and Y VALUES

Abstract— Pyrene fluorescence spectra have been run in 62 solvents of widely differing solvent polarity. As has been noted previously, the intensity ratio of the first (the 0–0 band) and third bands in vibronic fine structure of these spectra are very sensitive to solvent polarity. These I ₁/ I ₃ values, however, are not sensitive to hydrogen bonding aspects of solvent-solute interactions. Correlations are reported with Winstein's Y values and with Dimrotb's E _T values. On this basis the I ₁/ I ₃ values for pyrene fluorescence are suggested as the basis for a new empirical scale of solvent polarity, called the Py scale, which offers certain conveniences over other scales of solvent polarity.     

A REVERSIBLE PHOTOCHEMICAL REACTION OF CHLOROPHYLL a WITH I2

Abstract— Chlorophyll a and I₂ form a 1:1 addition compound, which exhibits a strong absorption maximum at about 360 nm. When dissolved in anaerobic methanol, this compound can be reversibly bleached by illumination with red light. Prolonged illumination at low temperatures (˜– 75°C) or intense illumination at room temperature reduces the height of the red absorp tion maximum to about half of its normal value. Since the thermal back reaction is negligibly slow at – 75°C, presumably the chlorophyll-I₂ complex is converted completely to its bleached form, when illuminated under those conditions. This assumption leads to a simple mechanism which is consistent with the experimental data.