REEVALUATION OF THE SPECTRAL AND KINETIC PROPERTIES OF HO2 AND O2- FREE RADICALS

Abstract— The spectra and molar absorbances of the HO₂ and O₂^- free radicals have been redetermined in aqueous formate solutions by pulse and stopped-flow radiolysis as well as by ⁶⁰Co gamma-ray studies. The extinction coefficients at the corresponding maxima and 23°C are ²²⁵= 1400 ± 80 M ^-1 cm^-1 and ²²⁵= 2350 ± 120 M ^-1 cm^-1 respectively. Reevaluation of earlier published rate data in terms of the new extinction coefficients yielded the following rate constants for the spontaneous decay of HO₂ and O₂^-: K _Ho2+HO2= (8.60 ± 0.62) × 10⁵ M ^-1 s^-1; K _Ho2+O2-= (1.02 ± 0.49) × 10⁸ M ^-1 s^-1; K _Ho2+O2- < 0.35 M ^-1 s^-1. For the equilibrium HO₂→ O₂^-+ H⁺ the dissociation constant is K _Ho2= (2.05 ± 0.39) × 10^-5 M or p K _HO2= 4.69 ± 0.08. G (O₂^-) has been evaluated as a function of formate concentration.     

DEPENDENCE OF Pfr/Ptot-RATIOS ON LIGHT QUALITY and LIGHT QUANTITY

Abstract— Not only the spectral distribution of the light source determines the relative proportion of phytochrome in the P_fr(P_r) form, the P_fr/P_tot-ratio also depends strongly on the fluence rate of the irradiation. This dependence has been observed in the cotyledons of etiolated mustard seedlings for blue light of fluence rates below 20 Wm_-2. It has also been observed for white light and seems to be a characteristic of the phytochrome system resulting from the involvement of phytochrome thermal reactions as well as P_r P_fr photoconversions. The fluence rate dependence of P_fr/P_tot-ratios can be used to analyze the characteristic transformations of the phytochrome system. Phototransformations together with a fast thermal transformation (τ_½⋍ 3min) are consistent with the results obtained for blue and white light.     

ON THE PRIMARY PHOTOPROCESS OF 124-kdalton PHYTOCHROME

The photoreaction between P_τ and the first detectable intermediate, lumi-R, of 124-kdalton oat phytochrome has been investigated at low temperatures. The temperature dependence of the quantum yields of the photoreactions, P_τ to lumi-R and lumi-R to P_τ, has been determined. From measurements over a temperature range from 119 to 155 K, an activation barrier of 3.6 ± 0.5 kJ mol ₁ is found for the photoreaction of P_τ with 661-nm actinic light. A higher value (5.7 ± 0.7 kJ mol ^-1) is found for the photoreaction of lumi-R to P^τ. with 698-nm actinic light. Increased quantum yields are found in deuterated buffer solutions at low temperatures. The activation energies for deuterated phytochrome (3.2 ± 0.7 kJ mol^–1 for P_τ with 661-nm irradiation and 6.2 ± 1.2 kJ mol^-1 for lumi-R at 698-nm irradiation) are identical within the limits of error with those of protonated phytochrome. The lack of a deuterium effect for the activation energies favors the Z,E-isomerization rather than proton transfer or tautomerization for the chromophore photochemistry during P_τ⇄lumi-R conversion.     

A PHYTOCHROME STUDY USING TWO-LASER/TWO-COLOR FLASH PHOTOLYSIS: I700 IS A MANDATORY INTERMEDIATE IN THE PrPfr PHOTOTRANSFORMATION

Abstract— The phototransformation of native (124 kDa)oat phytochrome, P_r P_fr, Has been studied at 10C by two laser/ two-color flash photolysis. the overall P_rP_fr reaction yield did not vary with temperature within the range4–21C. Foloeing the excitation of P_r with a single 15 ns laser flash at 650nm, the formation of P_fr was quantitavely measured in a time-resolved experiment in the presence of a second 8 ns laser flash at 710 nm delayed from the initial flash. the second laser flash causes at 1.0 s after the initial laser flash a depletion of the uintermediate I₇₀₀ as welll as a reduction of the P_fr absorption at 730 nm. The depletion of I₇₀₀ correlates quantitavely with the reduction of P_fr formation. The absorpton spectra of I₇₀₀ and of the following intermendiate, I_bi, were calculated assuming that the amount of P_r, which is photoconverted by a single laser, equals the amount of P_fr formed.     

Recombinant Phytochrome of the Moss Ceratodon purpureus: Heterologous Expression and Kinetic Analysis of Pr→ Pfr Conversion

Abstract— The phytochrome-encoding gene Cerpu;PHY;2 ( CP2 ) of the moss Ceratodon purpureus was heterologously expressed in Saccharomyces cerevisiae as a polyhistidine-tagged apoprotein and assembled with phytochromobilin (P φ B) and phycocyanobilin (PCB). Nickel-affinity chromatography yielded a protein fraction containing approximately 80% phytochrome. The holoproteins showed photoreversibility with both chromophores. Difference spectra gave maxima at 644/716 nm (red-absorbing phytochrome [P_r]) far-red-absorbing phytochrome [P_fr]) for the PCB adduct, and 659/724 nm for the PφB-adduct, the latter in close agreement with values for phytochrome extracted from Ceratodon itself, implying that PφB is the native chromophore in this moss species. Immunoblots stained with the antiphytochrome antibody APC1 showed that the recombinant phytochrome had the same molecular size as phytochrome from Ceratodon extracts. Further, the mobility of recombinant CP2 holophyto-chrome on native size-exclusion chromatography was similar to that of native oat phytochrome, implying that CP2 forms a dimer. Kinetics of absorbance changes during the P_r→ P_fr photoconversion of the PCB adduct, monitored between 620 and 740 nm in the microsecond range, revealed the rapid formation of a red-shifted intermediate (I₇₀o)> decaying with a time constant of - 110 u.s. This is similar to the behavior of phytochromes from higher plants when assembled with the same chromophore. When following the formation of the P_fr state, two major processes were identified (with time constants of 3 and 18 ms) that are followed by slow reactions in the range of 166 ms and 8 s, respectively, albeit with very small amplitudes.     

HO2 ELIMINATION FROM α-HYDROXYALKYLPEROXYL RADICALS IN AQUEOUS SOLUTION

Abstract— In aqueous solutions α-hydroxyalkylperoxyl radicals undergo a spontaneous and a base catalysed HO₂ elimination. From kinetic deuterium isotope effects, temperature dependence, and the influence of solvent polarity it was concluded that the spontaneous reaction occurs via an HO₂ elimination followed by the dissociation of the latter into H⁺ and O₂^-. The rate constant of the spontaneous HO₂ elimination increases with increasing methyl substitution in α-position ( k (CH₂(OH)O₂) < 10s^-1 k (CH₃CH(OH)O₂) = 52s^-1 k ((CH₃)₂C(OH)O₂) = 665 s^-1). The OH^- catalysed reaction is somewhat below diffusion controlled. The mixture of peroxyl radicals derived from polyhydric alcohols eliminate HO₂ at two different rates. Possible reasons for this behaviour are discussed. The mixture of the six peroxyl radicals derived from d -glucose are observed to eliminate HO₂ with at least three different rates. The fastest rate is attributed to the HO₂ elimination from the peroxyl radical at C-l ( k > 7000s^-1). Because of the HO₂ eliminations the peroxyl radicals derived from d -glucose do not undergo a chain reaction in contrast to peroxyl radicals not containing an α-OH group. In competition with the first order elimination reactions the α-hydroxylalkylperoxyl radicals undergo a bimolecular decay. These reactions are briefly discussed.     

THE ABSOLUTE VALUE OF THE REACTION RATE CONSTANT OF BILIRUBIN WITH SINGLET OXYGEN IN D2O

Abstract— The chemical reaction rate constant of bilirubin with singlet oxygen in basic aqueous solution has been redetermined to be 3.5 × 10⁸ M^-1 s^-1 by a competitive technique using a 1,3-diphenylisobenzofuran in sodium dodecyl sulfate micelles. Bilirubin also physically quenches a singlet oxygen with a rate constant of 9 × 10⁸ M ^-1 s^-1. The lifetime of singlet oxygen in D₂O solution has been determined to be 35 μ s . The absorption cross-section for the molecular oxygen ³∑_g-→¹δ _g + 1 v electronic transition at 1.06μn in aqueous solution is unexpectedly larger than the gas paase cross-section.     

CALCULATED RATES AND EQUILIBRIA OF THERMAL INTERCONVERSION OF TRIPLET (3g-) AND SINGLET (1Δg and g+) MOLECULAR OXYGEN

Abstract— From spectroscopic data and rate constants in the literature, equilibrium constants and rates of thermal formation of singlet oxygen (¹Δ_g and ¹Σ_g⁺) were calculated for a number of conditions. For the gas phase we estimate K _eq(¹Δ_g³Σ_g^-) = 1.67 exp(-94.31 KJ/RT) and K _eq(¹Σ_g⁺/³Σ_g^-) = 0.33 exp(-157.0 KJ/RT). The calculated rate constants for the ³Σ_g⁺→¹Δ_g transition of O₂ at 25°C varied from 2.5 × 10^-11 s^-1 in water to 4.8 × 10^-16 s^-1 in air, assuming equal solvent interactions with the ground and excited states. Physical quenchers for singlet oxygen are expected to be catalysts for its thermal formation. Equations are presented which allow one to estimate whether such catalysis by quenchers will result in a pro-oxidant effect.     

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.     

CHARACTERIZATION OF A MICROSECOND INTERMEDIATE IN THE LASER FLASH PHOTOLYSIS OF SMALL PHYTOCHROME FROM OAT

Abstract— Irradiation of small phytochrome from oat in its P_r form with 15 ns laser pulses of different wavelengths(605–655 nm) gave rise to a difference absorption with maxima at 400 and 685 nm for the first detectable transient. Bleaching of a 660 nm band was observed, non-recuperable up to 1 ms. The transient absorption has a lifetime of 70±15 μs at 273 K. The transient is tentatively identified as lumi-R and the conformation of its chromophore is postulated to be more extended than that of P_r. A deviation from the exponential decay of the lumi-R absorption at 284 and 300 K and the lack of observable enhancement of the far-red absorption within 1 ms are interpreted in terms of the appearance of still other intermediates on this time scale between lumi-R and P_fr phytochrome.     

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.     

Dynamics of Flavin in Flavocytochrome b2: A Fluorescence Study

Abstract— The dynamics of the flavin bound to the flavocytochrome b₂ from Hansenula anomala were studied by fluorescence intensity quenching and quenching emission anisotropy with iodide. The fluorescence intensity of bound flavin is decreased 13-fold as compared to the free molecule. The remaining fluorescence decays with two lifetimes equal to 0.963 ± 0.040 and 4.635 ± 0.008 ns and fractional intensities of 0.036 ± 0.002 and 0.964 ± 0.002, respectively. The bimolecular diffusion constant was found to be 3.33 × 10⁹ M ^-1 s^-1 when the flavin is bound to the enzyme and 8.3 × 10⁹ Mv s^-1 when the flavin is free in solution. Thus, the flavin in flavocytochrome b₂ is accessible to the solvent, but the amino acid residues of the binding site inhibit the diffusion of iodide. The rotational correlation time of bound flavin was found to be 2.015 ± 0.365 ns, a value higher than that (155 ps) of free flavin in solution. Our results are discussed on the basis of local dynamics of the flavin.     

THE PHOTOPHYSICAL PROPERTIES OF SOME C15 LUPINE ALKALOIDS:

Abstract Sparteine, the tetracyclic saturated amine alkaloid, is highly fluorescent in n-hexane solution (Φ_f= 0.64; ζ= 63 ns) and has a large Stokes shift [λ_max (abs) = 203 nm; λ_max (fluor) = 325 nm]. Its isomer, α-isosparteine, has similar properties: Φ_f= 0.55; ζ_f= 50 ns; λ_max (fluor) = 338 nm. Oxidized derivatives, such as lupanine, thermopsine, and α-diplospartyrine, are weakly fluorescent. Based on a comparison with spectroscopic and photophysical properties of the monoamine, quinolizidine, it is concluded that the excitation energy is delocalized over the two N-atoms in starteine and a-isosparteine. The self quenching rate constant for sparteine, ca. 1 times 10⁷M^-1 s^-1, is about 100 times smaller than that for quinolizidine, consistent with excited state derealization. The significant fluorescence quenching in lupanine is rationalized by the fact that N-methyl-2-piperidone mfe/molecularly quenches the fluorescence of quinolizidine at nearly the diffusion controlled rate in -hexane. Comparisons are made with the fluorescence properties of other diamines such as N, N'-dimethylbispidine and N, N'-disubstituted piperazines.     

124-kDa PHYTOCHROME IN MODEL MEMBRANE SYSTEMS: STUDIES OF THE Ii700 INTERMEDIATES WITH THE PROTEIN COVALENTLY BOUND TO PREFORMED LIPOSOMES

Abstract— We have described the covalent binding of 124-kDa oat phytochrome to large unilamellar liposomes composed of either dioleoyl phosphatidylcholine or dipalmkoyl phosphatidylcholine or soybean lecithin, without affecting the photochromic properties of the protein. These phytochrome-liposome systems have now been studied by laser flash photolysis. The liposomes, independent of their membrane rigidity (liquid-crystal vs gel-like phase), do not influence the ratio and reactivity of the two primary photoproducts, Iⁱ₇₀₀- of the red absorbing form of phytochrome, P_l Thus, the lifetimes of the Iⁱ₇₀₀ intermediates and the activation parameters associated with Iⁱ₇₀₀Iⁱ_bl are the same as those measured for nonbound phytochrome in buffer solution. The temperature increase from about 273 K. to 297 K lowers the population of the shorter-lived Iⁱ₇₀₀ intermediate to the same extent both in the liposome-P_l and in nonbound P_l, whereas it does not affect the relative population of the Iⁱ₇₀₀ intermediates from non-bound P_l in the presence of 25% ethylene glycol added to the buffer solution (ionic strength 0.17).     

ASCORBIC ACID AS A PHOTOOXIDATION INHIBITOR

Abstract— The rate constant for total quenching of singlet oxygen by ascorbic acid has been determined using the inhibition of the bleaching of 9, 10-dimethylanthracene by AA in pyridine. The rate constant was 8.4 × 10^-6 M ^-1 s^-1, as determined photochemically, and 1.06 10^-7 M ^-1 s^-1 as determined in a dark reaction.     

RATE CONSTANTS FOR INTERACTION OF 1O21Δg) WITH AZIDE ION IN WATER

Abstract— The rate constant for quenching of ¹O₂ by azide ion in water was determined to be (5.0 ± 0.4) × 10⁸ M ⁻¹ s⁻¹ using a variety of sensitizers (including humic acids) and ¹O₂ acceptors. The apparent second-order rate constant decreases with pH below pH 5.5 in accordance with the protonation of azide ion to form hydrazoic acid (p K _a= 4.6). Quenching by hydrazoic acid is at least 2 orders of magnitude slower than by azide ion. Greater than 99% of all interactions between ¹O₂ and azide ion involve physical quenching rather than chemical reaction. Humic acid triplets are not significantly quenched by azide ion at concentrations less than 2 m M , allowing azide ion quenching to be used as a diagnostic test for the intermediacy of ¹O₂ in photosensitized oxidations in natural surface waters.     

AN ANALYSIS OF PHYTOCHROME-MEDIATED THRESHOLD CONTROL OF HYPOCOTYL GROWTH IN MUSTARD (Sinapis alba L.) SEEDLINGS†

Hypocotyl elongation in mustard (Sinapis alba L.) seedlings is known to be controlled by phytochrome (P_fr) through a threshold response. This phytochrome-mediated threshold response was studied in detail with the following results: (i) The P_fr threshold value required to suppress hypocotyl growth is much lower (0.03% P_rr, based on total phytochrome in the hypocotyl at 36 h after sowing = 100%) than those threshold valued observed previously in threshold control by hook phytochrome of appearance of 'potential capacity for photophosphorylation' and lipoxygenase appearance in the mustard cotyledons (1.25% P_tr, based on total phytochrome in the hypocotyl at 36 h after sowing = 100%). This probably explains why hypocotyl elongation is so extremely sensitive to light, (ii) The P_fr threshold value controlling hypocotyl growth is a system constant, independent of total phytochrome content, developmental age and actual growth rate, (iii) Threshold control of hypocotyl elongation is unaffected by the removal of the cotyledons and half of the hook. However, removal of the whole hook totally eliminates any light control over the residual hypocotyl growth, (iv) After termination of the threshold control, the hypocotyl growth rate immediately returns to precisely that found in untreated dark control even though the partial growth rates of the different parts of the hypocotyl are quite different, relative to their dark controls. Obviously, the organ grows as an integrated unit.
It is concluded that the all-or-none threshold control over hypocotyl growth is exerted from the plumular hook. It appears that the hook can send off phytochrome all-or-none signals in both directions, to the cotyledons and to the hypocotyl.     

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.     

USE OF TRIS (2,2'-BIPYRIDINE) RUTHENIUM(II) DICATION IN A NOVEL METHOD FOR THE DETERMINATION OF SINGLET OXYGEN REACTION RATES: APPLICATION TO STUDIES OF GELATIN

Abstract— A novel method for the determination of singlet oxygen reaction rate constants is described and applied to studies of cyclohexadiene in methanol and gelatins in H₂O and D₂O. The technique uses tris (2,2'-bipyridine) ruthenium(II) dication (Ru(bipy)₃₂₊) as both singlet oxygen sensitizer and in situ oxygen concentration monitor during irradiation of sealed samples. Because of the high efficiency with which the luminescence of Ru(bipy)₃2+* can be detected, and the fact that emission lifetimes are used, the method offers some advantages over those previously described. The advantages and disadvantages of the method are discussed. A rate constant of 2.1 (±0.3) x 10⁶ mol^-1 dm³ s^-1 has been determined for the reaction of ¹O₂ with cyclohexadiene in methanol. For two different photographic gelatins the sums of reaction and quenching rate constants are 2.0 (±0.4) x 10⁶ and 3.1 (±2.0) x 10⁵ mol^-1 dm³ s^-1; for swine skin gelatin this value is 3.9 (±2.4) × 10⁵ mol^-1 dm³ s^-1. Chemical reaction, rather than physical quenching, is the dominant reaction route for gelatins and ¹O₂.     

THE DOSE RESPONSE CURVE IN PHYTOCHROME-MEDIATED ANTHOCYANIN SYNTHESIS IN THE MUSTARD SEEDLING

Abstract —The dose response curve for light (phytochrome)-induced anthocyanin synthesis was determined in the mustard seedling. The curve gives the amount of anthocyanin (A) synthesized within 24 h as a function of the amount of P_fr* produced by a brief light pulse. The [P_fr] response curve is composed of two linear parts with very different slopes ( a _1,2) connected by a relatively narrow transient range (curved segment). The [P_fr] response curve extrapolates precisely through zero [P_fr]. The reciprocity law is valid over the whole range investigated (up to 320 s of irradiation). It is concluded that the initial (or primary) reaction of P_fr (P_fr+ X → P_frX) does not involve any significant cooperativity in the case of phytochrome-mediated anthocyanin synthesis. It is speculated that the linear parts of the [P_fr] response curve truly reflect the mode of phytochrome action ( A = a _1,2 [P_fr]; X does not come into play since it is not rate limiting) whereas the curved segment represents a transition of the reaction matrix of P_fr. The large difference between a₁ and a₂ seems to indicate that the physiological effectiveness of a given amount of P_fr (or P_frX) is determined by [P_fr] through a P_fr-induced change in the reaction matrix.