INTERMEDIATES IN THE PHOTOCONVERSION OF FUNCTIONAL PHYTOCHROME IN FERN SPORES OF Dryopteris-I DEMONSTRATION AND QUANTITATIVE CHARACTERIZATION OF THE PHOTOCHROMIC SYSTEM Pr⇔ Ii700USING NANOSECOND-LASER PULSES*,†

Abstract— Spores of Dryopteris paleacea and D. filix-mas are positively photoblastic with an optimum in the action spectrum around 665 nm. Light is perceived by phytochrome and the relationship between germination and mole fraction of the far-red-absorbing form of this pigment, P_fr, was investigated with saturating irradiations between 662 and 747 nm under low-fluence-rate conditions. These control irradiations establish a proportion of the total phytochrome, P,_tot, as P_fr with P_fr/P_tot–φ at equilibrium. These φ -values were calculated according to data for native oat phytochrome (Kelly and Lagarias, 1985, Biochemistry 24, 6003) and the spectral characteristics of the interference filters. With this method a linear relationship could be found between φ and germination from 2 to 70% for D. paleacea and from 2 to 90% for D. filix-mas, if probit germination was plotted vs probit φ This correlation formed the basis of investigating the phytochrome photoconversion by dye-laser pulses of 380 ± 30 ns under high-fluence-rate conditions, and thus to test quantitatively the impact of the photoreversibility of intermediate reactions of the photoconversion and the red-absorbing form of phytochrome, P_fr on the final P_fr-level. Spore germination was initiated by a single-laser pulse in the range from 592 to 700 nm. The most effective wavelengths were 649 and 660 nm in both species, and at saturation maximal germination (ca. 50%) was obtained from 592 to 665 nm for D. paleacea or ca. 60% germination from 592 to 670 nm for D. filix-mas. Both saturation levels correspond to a ø-value between 0.40 and 0.45. This significantly diminished photoconversion is a consequence of the high-fluence-rate conditions during the laser pulse which establishes the photochromic system between P_r and a set of very early intermediates, Iⁱ_700, (= P_r? Iⁱ₇₀₀). This system can be described by the extinction coefficients of P_r and the intermediates Iⁱ₇₀₀, and by the quantum yields, 4,φ for the forward and reverse reactions as φ If φ is calculated, assuming a quantum yield of 1:1 for both reactions and with the extinction coefficients of P_r and Iⁱ_7(l() (= lumi-R) given by Eilfeld and Riidiger (1985, Z. Naturforsch. 40c , 109), significantly higher values are calculated for / as compared to φ found in the control experiments. These results can be explained either: (i) with a quantum yield ratio φ_pr-φ1700: φ_1700φpr=1:1 and an assumed additional dark reaction leading from Iⁱ₇₀₀ or later intermediates back to P_r: or (ii) with a quantum yield ratio φ_{pr φ 1700}: φ_{1700 φpr}=1:2. In this case all Iⁱ₇₀₀ have to relax to P_fr. In this case all Iⁱ₇₀₀ have to relax to P_fr.     

Primary Quantum Yield and Volume Change of Phytochrome-A Phototransformation Determined by Laser-Induced Optoacoustic Spectroscopy*

The primary quantum yield, Φ₁₇₀₀, for the photoconver-sion of the red-absorbing form of phytochrome, P_r, to the set of primary intermediates, Iⁱ₇₀₀, was redetermined by laser-induced optoacoustic spectroscopy at very low excitation laser fluences. The Iⁱ₇₀₀ value obtained is in the range of Φ_Pfr reported for the complete phototransformation P_r→ P_fr (J. M. Kelly and C. Lagarias, 1985, Biochemistry 24 , 4003–6010). An energy level of ca 150 kJ/ mol was found for the intermediates Iⁱ₇₀₀, i.e. ca 85% of the 0–0 level of P_r. Furthermore, a molecular expansion of 7 mL/mol (equivalent to 11 ų/molecule) was determined for the P_r→ Iⁱ₇₀₀ transformation. It reflects the protein reorganization induced by the geometrical pho-toisomerization of the chromophore, which results in changes of bonding interactions, in particular between the chromophore and its protein surrounding.     

THE KINETICS OF THE EARLY STAGES OF THE PHYTOCHROME PHOTOTRANSFORMATION Pr→ Pfr. A COMPARATIVE STUDY OF SMALL (60 kDalton) and NATIVE (124 kDalton) PHYTOCHROMES FROM OAT

A comparative study of the decay kinetics of photogenerated transients from small (60 kDalton) and native (124 kDalton) oat phytochrome in the red-absorbing form (P_r) in phosphate buffer containing 5 mM ethylenediamine tetraacetic acid, pH 7.8, (PB) and in PB containing 20% ethylene glycol, has been carried out in the temperature range 275–298 K. The analysis confirmed that at least two primary photoproducts, intermediates Iⁱ₇₀₀s and Iⁱ_7oo are formed from P_r. The kinetic parameters, as observed in PB at 695 nm and 275 K, are similar for the I₇₀₀ intermediates of both small and native phytochrome. Namely, the lifetimes are about 21 μs (component percentages 38%) for the I Iⁱ₇₀₀s and about 200 μ.s (62%) for the Iⁱ₇₀₀S- Arrhenius preexponential factors (A) of about 10¹⁶ and 10¹⁵ s-¹and activation energies of about 61 and 56 kJ/mol were measured for the absorbance decays of the I₇₀₀S of small and native phytochrome, respectively. The kinetic data favour parallel paths for the formation of the Iⁱ₇₀₀s from P_r, and the activation parameters indicate that the primary photoreactions of the transformation from P_r to the far-red-absorbing form are restricted to the chromophore within the protein. Moreover, the relatively modest temperature dependence of the lifetimes of the Iⁱ₇₀₀^S from small and native P_r supports the working hypothesis that the ground state reactions to the Iⁱ_bl, intermediates–although somewhat influenced by the polypeptide fragment that is removed upon degradation of native to small P_r–are localized to the chromophore, as is most probably the case also for the primary photoreactions. The effect of the addition of 20% ethylene glycol on the pre-exponential factors of the time-dependent decay functions is discussed in similar terms of the early stages of the phototransformation.     

THE PHOTOPHYSICS OF BONELLIN: A CHLORIN FOUND IN MARINE ANIMALS

The photophysical properties of bonellin, a free-base chlorin, were studied in ethanolic solution. For the singlet excited state the following data were determined: an energy level, E^B_S= 187 ± 2kJ mol^-1, a lifetime, τ_f= 6.3± 0.1ns at 298 K, and fluorescence quantum yields, φ_r= 0.07 ± 0.02 (298 K) and 0.20 ± 0.04 (77 K). The S₁→ T intersystem crossing quantum yield was φ_isc= 0.85 ± 0.1. No phosphorescence was observed at 298 K and 77 K. Based on quenching experiments the triplet state energy level was determined to be E^B_T= 180 ± 20 kJ mol^-1. A unimolecular decay rate constant, k₁= (2.3 ± 0.5)· 10³ s^-1 at room temperature, and a molar absorption coefficient, ε^T₄₄₃= 9500 ± 500 M^-1 cm^-1, were obtained for the triplet state. This species was quenched by O₂ with ko₂= (1.7 ±0.3)· 10⁸M^-1 s^-1, and by benzoquinone with k_q= (5.2 ± 0.3)-10⁹M^-1 s^-1. The latter value, as well as the high value determined for the triplet annihilation rate constant, k₂= (2 ± 0.5)· 10⁹M^-1 s^-1, might reflect an electron transfer mechanism. Copper bonellin had a shorter triplet lifetime (>20 ns), which offers a possible explanation for its lack of photodynamic action.     

KINETICS OF Pfr APPEARANCE IN Amaranthus caudatus‡

Abstract— The kinetics of the far-red absorbing form of phytochrome (P_fr) appearance from intermediates in the pathway from the red absorbing form of phytochrome (P_r) to P_fr that accumulate under high fluence rate white light have been investigated in 3-day old dark grown Amaranthus caudatus seedlings. The appearance of P_(r after a 5 s white light pulse was measured over the temperature range -8 to 25^°C in samples flushed with O₂ or N₂. Over the whole temperature range under anaerobic conditions the kinetics of the slowest component of P_fr appearance are faster than in the presence of O₂. Arrhenius plots are linear over this temperature range and indicate the activation energy for the slowest component of P_fr appearance is 44.05 ± 1.97 kJ mol^?1 for O₂ and 53.69 ± 4.86 kJ mol^?1 for N₂.     

THE pKa OF DIPROTONATED SEMIMETHYLENE BLUE, IN 5% ETHANOL AND 50% ACETONITRILE AQUEOUS SOLUTIONS

Abstract— Semimethylene blue was generated by reductive quenching of triplet methylene blue, ³MBH²⁺, with diphenylamine at pH 0.62–3.4. A Q-switched ruby laser flash-photolysis-kinetic spectro-photometric apparatus was used to characterize the absorption spectrum of semimethylene blue from 350 to 900 nm and a number of physical constants at 25°C with μ= 0.4 M and Cl^? as the anion. The specific rate of quenching of ³MBH²⁺ by DPA is 2.8 × 10⁹M^?1 s^?1 in 5% EtOH-95% water and 1.2×10⁹M^?1 s^?1 in 50 v/v% aq. CH₃CN. Corresponding efficiencies of net electron transfer are, respectively, 0.15 and 0.62. Spectral characteristics in 5% EtOH are, for MBH²₂±, λ_max= 375 nm, ε₃₇₅= 9000 M^?1 cm^?1; λ_max= 880 nm, ε₈₈₀= 12700 M^?1 cm^?1; for MBH±, λ_max= 410 nm, ε₄₁₀= 9800 M^?1 cm^?1, λ_max= 880 nm, ε₈₈₀= 33000 M^?1 cm^?1; for MBH± in 50 v/v% AN, λ_max= 400 nm, ε₄₀₀= 11000 M^?1 cm^?1 and λ_max= 880 nm,ε₈₈₀= 39000 M^?1 cm^?1. The pK_a of MBH²₂ε calculated from the pH dependence of the absorption spectrum is 1.86 × 0.04 in 5% EtOH and 1.15 in 50 v/v% AN. Rate constants, k_decay, for reaction DPAH±⁺ with MBH²₂ε and MBH± in 5% EtOH are, respectively, 3.9 × 10⁹ and 9.5 × 10⁹M^?1 s^?1. The value of pK_a of MBH²₂ε calculated from the dependence of k_decay on pH is 1.75 in 5% EtOH.     

THE PHOTOEXCITED TRIPLET STATE OF TETRAPHENYL CHLORIN, MAGNESIUM TETRAPHENYL PORPHYRIN AND WHOLE CELLS OF CHLAMYDOMONAS REINHARDI. A LIGHT MODULATION-EPR STUDY

Abstract— The photoexcited triplet states of frozen solutions of tetraphenyl chlorin (TPC), magnesium tetraphenyl porphyrin (MgTPP) and whole cells of Chlamydomonas reinhardi have been studied by light modulation-EPR spectroscopy. The porphyrins were chosen to be studied as model compounds for chlorophyll molecules, From EPR spectra the zero field splitting parameters (ZFS) were calculated. For TPC, |D| = 0.0364 ± 0.0002 cm^-1, |E| = 0.0063 ± 0.0002 cm^-1. For MgTPP, |D| = 0.0310 ± 0.0002 cm^-1. For chloroplasts, |D| = 0.0280 ± 0.0004 cm^-1, |E| = 0.0032 ± 0.0004 cm^-1. In all compounds studied, except MgTPP, electron spin polarization (ESP) was observed. From the analysis of the kinetic curves at each canonical orientation we evaluated the spin lattice relaxation rate W, the depopulation rate constants k_p, and the ratio between the population rate constants, A_p, at zero magnetic field. For TPC in ethanol-toluene (5:1) k_x= (0.70 ± 0.10) × 10³ s^-1, k_y= (0.40 ± 0.07) × 10³ s^-1, k_x= (0.24 ± 0.05) × 10³ s^-1; A_x:A_y:A_z? 1.0:0.6:0.4; W= (2.60 ± 0.40) × 10³ s^-1. For MgTPP, only the total decay rate constant, k_T, was calculated: (1.5 ± 0.2) × 10 s^-1 in n-octane and (4.8 ± 0.8) × 10 s^-1 in ethanol. The results for TPC and MgTPP are compared to those reported previously for chlorophyll. It is concluded that the dynamics of the photoexcited triplet state in chlorophylls are mainly governed by the chlorin macrocycle. From the EPR spectrum and ZFS parameters of chloroplasts, we propose that both chlorophyll a and chlorophyll b are the main constituents of the EPR spectrum. From the analysis of the kinetic curves we obtain separately the kinetic parameters for chlorophylls a and b, k^a_x= (1.30 ± 0.20) × 10³ s^-1, k^a_y;= (0.85 ± 0.15) × 10³ s^-1k^a_x= (0.32 ± 0.05) × 10³ s^-1; A^a_x:A^a_y:A^a_z? 1.0:0.7:0.2; W^a= (1.20 ± 0.20) × 10³ s^-1; k^b_x= (0.56 ± 0.09) × 10³ s^-1, k^b_y= (0.30 ± 0.04) × 10³ s^-1, k^b_z= (0.06 ± 0.01) × 10³ s^-1; A^b_x:A^b_y:A^b_x? 1.0:0.6:0.1; W^b= (5.00 ± 0.80) × 10³ s^-1. These results are very close to those found separately for chlorophyll a and chlorophyll b oligomers in vitro.     

A PHYTOCHROME PHOTOTRANSFORMATION STUDY USING TWO-LASER/TWO-COLOR FLASH PHOTOLYSIS: ANALYSIS OF THE DECAY MECHANISM OF I700*

The mechanism of I₇₀₀ decay, representing an early event in the phytochrome P_r→ P_fr phototransformation, was reanalyzed in the microsecond range by conventional laser flash photolysis as well as by two-laser/two-color flash photolysis. Three kinetic models that might describe the I₇₀₀ decay mechanism following P_r excitation were considered: a parallel, a sequential, and an equilibrium model. These models were used to mathematically simulate both the one- and two-laser flash experiments in an effort to select the model best describing the I₇₀₀ decay. The sequential model could be excluded already on the basis of the one-laser flash photolysis results alone. Discussion of the two-laser/two-color flash rcsults in the context of the equilibrium and the parallel models is presented.     

ANALYSIS OF Pfr DESTRUCTION IN AMARANTHUS CAUDATUS L EVIDENCE FOR TWO POOLS OF PHYTOCHROME*

Abstract— Kinetics of the destruction of the far red absorbing form of phytochrome (P_fr), measured by in vivo spectroscopy, show two phases: after a saturating red light pulse, rapid first order decay results in the loss of most, but not all, of the detectable P_rr; decay of the rest is much slower. The concentration of the more stable P_fr is positively correlated to the concentration of the total P_fr established at time zero. The linear relationship between total and ‘stable’ P_fr exludes the existence of a threshold level of P_fr for fast destruction. Photoconversion of the P_r (red absorbing form of phytochrome) present during the slow decay, by exposure to a second light pulse, is followed by fast destruction of most of the newly formed P,_r, whereas some P_fr formed by the first pulse still remains. The experiment suggests that not all P_fr molecules are accessible to the same destruction mechanism, i.e. there are two populations of P_fI.     

ACTION SPECTRA OF PHYTOCHROME IN VIVO*

Abstract— A method is described to determine spectral properties of phytochrome in vivo. For photochrome in 7-day-old dark-grown Cucurbita pepo L. seedlings the mole fraction of the far-red-absorbing form (P_fr) present at photoequilibrium at 664 nm was found to be 0.76 ± 0.02 in vivo. Based on reflectance measurements, the photon fluence rate just below the surface of the cotyledons was calculated. Local rates of photoconversion for known local fluence rates were measured across cotyledons after non-saturating irradiations with wavelengths between 544 and 781 nm and in situ molar photoconversion coefficients were obtained. In contrast to purified oat phytochrome, the in situ molar photoconversion coefficients for P_fr show a strong shoulder between 660 and 700 nm. The maximum of P_fr absorption is at 726 nm. An isosbestic point of phytochrome is found at 686 nm. The mole fraction of P_fr present at photoequilibrium with 686 nm light is 0.58. The ratio of photoconversion quantum yields (that for P_r→ P_fr divided by that for P_fr→ P_r) is 1.38 ± 0.06.     

PHOTOPHYSICS OF THE FLUORESCENT Ca2+ INDICATOR QUIN-2

The photophysics of the complex forming reaction between Quin-2 and Ca²⁺ were investigated using steady-state and time-resolved fluorescence measurements. The fluorescence decay traces were analyzed with global compartmental analysis yielding the following values for the rate constants at room temperature in aqueous solution with EGTA as Ca²⁺ buffer: k₀₁= 8.6 times 10⁸ s^?1, k₂₁= 1 times 10¹¹M^?1 s^?1, k₀₂= 8.8 times 10⁷ s^?1, k₁₂= 4 times 10⁴ s^?1. k₀₁ and k₀₂ denote the respective deactivation rate constants of the Ca²⁺ free and bound forms of Quin-2 in the excited state. The constant k₂₁ represents the second-order rate constant of binding of Ca²⁺ and Quin-2 in the excited state while k₁₂ is the first-order rate constant of dissociation of the excited Ca²⁺:Quin-2 complex. From the estimated values of k₁₂ and k₂₁ the dissociation constant K_d* in the excited state was calculated. It was found that pK_d* (6.4) is slightly smaller than pK_d (7.2). There was no interference of the excited-state complex forming reaction with the determination of K_d. Intracellular Ca²⁺ concentrations can thus accurately be determined from fluorometric measurements using Quin-2 as Ca²⁺ indicator.     

IRRADIATION-ENHANCED PHYTOCHROME PELLETABILITY IN AVENA: IN VIVO DEVELOPMENT OF A POTENTIAL TO PELLET AND THE ROLE OF Mg2+ IN ITS EXPRESSION IN VITRO

The enhanced phytochrome pelletability that results from in vivo irradiation of Avena shoots may be divided into two operationally defined sequential stages: the in vivo development of a “potential to pellet” and the “expression” of this potential in vitro. Kinetic studies confirm previous findings that the generation of this “potential to pellet” is a very rapid (complete in < 10 s, 25°C), genuinely intracellular process, itself photoreversibly induced by P_fr. In addition, it is shown that the sustained development of the “potential to pellet”, that proceeds in the dark at 0°C following a red pulse, requires P_fr continually in the cell over the entire development period. Far red light immediately terminates further development of the red-induced “potential” at any point during the development phase. No immediate reduction is observed, however, in that level of “potential pelletability” already attained at the time of the far red pulse. This indicates that the level of “potential pelletability” established in vivo is insensitive to the form of the pigment at extraction regardless of the level reached. “Expression” of the “potential to pellet” refers to the actual detection in homogenates of an enhanced physical association of phytochrome with pelletable material. Maximum “expression” requires the presence of a divalent cation in the medium during homogenization. Rapid posthomogenization addition of Mg²⁺ to Mg²⁺-free extracts sustains enhanced pelletability but with rapidly declining effectiveness over the fmt 1–2 min after extraction. The rate of decline is faster if the phytochrome is present as P_fr than as P_r in the homogenate. Neither these nor previous data permit a distinction to be made between (a) preservation by the cation of a pre-existing intracellular interaction, and (b) a Mg²⁺-mediated induction of an artifactual, in vitro association predetermined in the cell by a genuine phyto-chrome-controlled process. Various formalistic models are discussed in the context of these and other data.     

AN ATTEMPT TO LOCALIZE THE THRESHOLD REACTION IN PHYTOCHROME-MEDIATED CONTROL OF LIPOXYGENASE SYNTHESIS IN THE MUSTARD SEEDLING

Abstract —Synthesis* of the enzyme lipoxygenase (LOG)? in the cotyledons of the mustard seedling (Sinapis alba L.) is controlled by phytochrome (P_fr) through a threshold (all-or-none) mechanism. The data of the present paper confirm the previous assumption (Oelze-Karow and Mohr, 1973) that the primary reaction of P_fr (P_fr+ X → P_frX ? P_frX‘) is the site of the highly cooperative threshold reaction. Suppression of LOG synthesis depends on the presence of P_frX’. However, P_frX‘ is only stable above the threshold level of P_fr. If the level of P_fr is below the threshold, P_frX is stable, and no suppression of LOG synthesis occurs. As long as the level of P_fr remains below the threshold, no destruction of P_fr takes place. Destruction of P_fr occurs only as long as [P_fr]?is above the threshold level. Thus the simplest formulation of the actual threshold reaction in the LOG response is P_frX?_frX’ state at [P_fr] below threshold no P_fr destruction LOG synthesis suppressed state at [P_fr] above threshold P_fr destruction(¹k_d LOG synthesis unimpaired The reversible threshold reaction is thus an integral part of the “primary reaction” of P_fr occurring at the “matrix” specific for the LOG response. The data and conclusions on the LOG response are consistent with an “open phytochrome-receptor model” recently advanced by E. Schäfer (1975). The data are not consistent with the concept that a rapid dark reversion (P_fr→P_r) exists in dicotyledonous seedlings and that the degree of P_fr dark reversion strongly depends on the initial photostationary state, φ?, established by a saturating light pulse.     

FLUORESCENCE OF PHYTOCHROME IN THE CELLS OF DARK-GROWN PLANTS AND ITS CONNECTION WITH THE PHOTOTRANSFORMATIONS OF THE PIGMENT

Abstract Fluorescence of phytochrome is found in the cells of etiolated monocotyledonous and dicotyledonous plants. The red light-absorbing form of phytochrome (P_r) fluoresces at 77 K with a yield 0.3±0.1 and maxima at 672–673 nm and 684–686 nm in the excitation and emission spectra, respectively. The emission is characterized by the sharp temperature dependence of its intensity, its high (～ 40%) polarization, and the violation of the mirror symmetry rule. Connection of the fluorescence with P_r photoreactions is followed in the interval 77–293 K. A P, photoproduct, lumi-R, is fluorescent with maxima at 696 nm and 705 nm in the excitation and emission spectra; the far-red light absorbing form of phytochrome (P_fr) is practically nonfluorescent. Three isochromic emitting P_r species are present differing in their photochemical properties: P_r1 and P_r2 which phototransform irreversibly and reversibly at T 170 K into lumi-R, and lumi-R₂, respectively, and P_r3 which undergoes photoconversion only at T > 240 K. The activation energies of P_r2 and P_r3 photoreactions are evaluated to be 2.9–3.3 kJ/mol and 26 kJ/mol. Complex dynamics of changes of P_r fluorescence and of the extent of its decrease in the photoconversion P_r? P_fr in germinating pea and bean seeds suggests the existence of two P_r pools one of which is incapable of P_r? P_fr phototransformation. Thus, the developed fluorescent method of phytochrome assay and investigation in the cell revealing multiplicity of phytochrome states in vivo proves to be very sensitive (about 1 ng) and informative.     

STUDIES ON PHYTOCHROME PHOTOCONVERSIONS IN VITRO WITH LASER-INDUCED OPTOACOUSTIC SPECTROSCOPY*

After excitation at room temperature with a 15 ns dye laser pulse, phytochrome (60 kDalton) from etiolated oat seedlings was studied by optoacoustic spectroscopy, which records the heat emission caused by non-radiative deactivation processes of the photoexcited molecule. The action spectrum for the heat emitted by P_r deviates from the absorption spectrum around 610 and 695 nm, indicating the build-up of photoproduct(s) within 15 ns after excitation. It is proposed that the 695 nm product is identical with I₇₀₀ and lumi-R known to be the first intermediate on the P_r× P_fr pathway, and that the photochemical back reaction of I₇₀₀ to P_r occurs on the same time scale. About 90% of the absorbed light energy was lost by radiationless deactivation. Values for the rate constants of excited state deactivation of P_r and for the internal energy content difference of the primary photoproducts are calculated.     

Absolute rate constants for the reactions ofOHradicals withCH3CH2OH, CF2HCH2OHandCF3CH2OH

Summary Pulsed laser photolysis with resonance fluorescence monitoring of OH radicals was applied at T = 300±2 K to obtain the rate constants of k₁= (3.38±0.60)x10^-12, k₂= (2.52±0.44)x10^-13and k₃ = (1.06±0.30)x10^-13cm³molecule^-1s^-1with 2σprecision given for the overall reactions OH + CH₃CH₂OH (1), OH + CF₂HCH₂OH (2) and OH + CF₃CH₂OH (3), respectively. k₂is the first direct kinetic data for the reaction of OH radicals with CF₂HCH₂OH reported in the literature.</o:p>     

THE MECHANISM OF THE 9,10-DIBROMOANTHRACENE MEDIATED TRIPLET-SINGLET ENERGY TRANSFER IN THE TETRAMETHYL-1,2-DIOXETANE ENERGIZED RUBRENE LUMINESCENCE*

Abstract— The ternary chemiluminescent system consisting of tetramethyl-dioxetane (TMD), 9 ,10-dibromoanthracene (DBA) and rubrene (Ru) has been investigated in benzene solution and in polystyrene matrices. DBA has been found to mediate the energy transfer between excited triplet acetone (³K), generated from TMD, and rubrene resulting in the enhancement of the Ru emission and reduction in the DBA emission. A detailed kinetic analysis confirms that ca. 50% of the enhanced chemilumines-cence involves triplet-singlet (TS) energy transfer from ³K to DBA, followed by singlet-singlet (SS) energy transfer from DBA to Ru, the remainder ca. 50% being due to reabsorption of DBA fluorescence by rubrene. It is concluded that the TS energy transfer is of the resonance type, occurring with a rate of k^TS_K.DBA= (1.4 ± 0.4) ± 10⁹M^-1s^-1 and an efficiency of ø^TS_K.DBA= 0.3 ± 0.1. As expected, the SS energy transfer is also of the resonance type, taking place at comparable rates in benzene solution and in polystyrene matrices, is. k^SS_DBARU= 1.5 ± 10¹⁰ M^-1 and (2.2 ± 0.2) ± 10¹⁰M^-1s^-1, respectively.     

KINETICS OF FORMATION AND STABILITY CONSTANTS OF MONO AND BIS l-TYROSINE COMPLEXES OF Cu(II), Co(II), AND Ni(II)

Abstract

The kinetics and stability constants of l-tyrosine complexation with copper(II), cobalt(II) and nickel(II) have been studied in aqueous solution at 25° and ionic strength 0.1 M. The reactions are of the type M(HL)^(3-n)+ _n-1 + HL- ? M(HL)^(2-n)+_n(k_n, forward rate constant; k_-n, reverse rate constant); where M=Cu, Co or Ni, HL^? refers to the anionic form of the ligand in which the hydroxyl group is protonated, and n=1 or 2. The stability constants (K_n=k_n/k_-n) of the mono and bis complexes of Cu²⁺, Co²⁺ and Ni²⁺ with l-tyrosine, determined by potentiometric pH titration are: Cu²⁺, log K₁=7.90 ± 0.02, log K₂=7.27 ± 0.03; Co²⁺, log K₁=4.05 ± 0.02, log K₂=3.78 ± 0.04; Ni²⁺, log K₁=5.14 ± 0.02, log K₂=4.41 ± 0.01. Kinetic measurements were made using the temperature-jump relaxation technique. The rate constants are: Cu²⁺, k₁=(1.1 ± 0.1) × 10⁹ M ^?1 sec^?1, k_-1=(14 ± 3) sec^?1, k₂=(3.1 ± 0.6) × 10⁸ M ^?1 sec^?1, k^?2=(16 ± 4) sec^?1; Co²⁺, k₁=(1.3 ± 0.2) × 10⁶ M ^?1 sec^?1, k_-1=(1.1 ± 0.2) × 10² sec^?1, k₂=(1.5 ± 0.2) × 10⁶ M ^?1 sec^?1, k_-2=(2.5 ± 0.6) × 10² sec^?1; Ni²⁺, k₁=(1.4 ± 0.2) × 10⁴ M ^?1 sec^?1, k_-1=(0.10 ± 0.02) sec^?1, k₂=(2.4 ± 0.3) × 10⁴ M ^?1 sec^?1, k_-2=(0.94 ± 0.17) sec^?1. It is concluded that l-tyrosine substitution reactions are normal. The presence of the phenyl hydroxyl group in l-tyrosine has no primary detectable influence on the forward rate constant, while its influence on the reverse rate constant is partially attributed to substituent effects on the basicity of the amine terminus.     

Spin polarization in the lowest triplet state of chlorophyll

Chlorophyll-b in glassy solution has a spin-polarized lowest triplet state at and above 77 K. The magnitude of the effect is different for MTHF and ethanol as solvents, in contrast to what is found for the porphin free base. Chlorophyll-a does not exhibit spin-polarization under identical conditions as for chlorophyll-b. Zero-field parameters are found to be:chlorophyll-a (MTHF) D = (281 ± 6) × 10^?4 cm^?1; E = (39 ± 3) × 10^?4 cm^?1;chlorophyll-b (MTHF) D = (289 ± 4) × 10^?4 cm^?1; E = (49 ± 3) × 10^?4 cm^?1,From ESR signal kinetics it follows that for chlorophyll-b, population and depopulation mainly involve the spin level y?, describing a spin moving in a plane perpendicular to the molecular plane:P_y ? P_x ? P_z; k_x = 240 ± 40 s^?1; k_y = 600 ± 120 s^?1; k_z ? 75 s^?1,where P_i and k_i denote populating and decay rates. Thus, the kinetic scheme for the chlorophyll triplet is different from that of porphyrins with heavier metal ions, but very similar to that of the porphin free base. The spin-lattice relaxation time is found to be anisotropic and shorter than the decay rates of individual spin levels. Nevertheless, spin polarization can be observed, essentially because the ESR signal amplitude depends on population differences.     

Rate coefficients for reactions of OH radicals with CH3D,CH2D2, CHD3, and CD4

Fourier transform infrared (FTIR) smog chamber techniques were used to investigate the atmospheric chemistry of the isotopologues of methane. Relative rate measurements were performed to determine the kinetics of the reaction of the isotopologues of methane with OH radicals in cm³ molecule⁻¹ s⁻¹ units: k(CH₃D + OH) = (5.19 ± 0.90) × 10⁻¹⁵, k(CH₂D₂ + OH) = (4.11 ± 0.74) × 10⁻¹⁵, k(CHD₃ + OH) = (2.14 ± 0.43) × 10⁻¹⁵, and k(CD₄ + OH) = (1.17 ± 0.19) × 10⁻¹⁵ in 700 Torr of air diluent at 296 ± 2 K. Using the determined OH rate coefficients, the atmospheric lifetimes for CH_4–xD_x (x = 1–4) were estimated to be 6.1, 7.7, 14.8, and 27.0 years, respectively. The results are discussed in relation to previous measurements of these rate coefficients.