Flash Photolysis Methods Research Articles

Calcium and liver

Cells expand energy to lower the concentration of free calcium in the cytosol ([Ca2+]i) to a very low level. Extracellular Ca2+ entering via channels situated in the plasma membrane is expelled into the extracellular medium by a Ca(2+)-Mg(2+)-ATPase or by Na(+)-Ca2+ exchangers. The Ca2+ that enters the cell is sequestered, once inside the cytosol, by a Ca(2+)-Mg(2+)-ATPase, which concentrates Ca2+ in specialized domains of the endoplasmic reticulum. The nucleus and the mitochondria also concentrate Ca2+, but less efficiently. The stimulation of numerous receptors by hormones, growth factors and neurotransmitters coupled to GTP-binding proteins provokes a rapid increase in [Ca2+]i by mobilizing Ca2+ from intra- and extracellular compartments. Membrane coupling is ensured by the activation of a phospholipase C-beta, which hydrolyses a doubly phosphorylated phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). The inositol (1,4,5)-trisphosphate (InsP3) consequently formed binds to a receptor consisting in 4 homologous of 250 kDa each. The InsP3 receptor has been localized to a specialized region, rich in Ca2+, of the endoplasmic reticulum. The receptor has been purified and its sequence obtained. Reincorporated into planar bilayers, it displays the properties of a channel. In the cell, opening of the InsP3 receptor-channel provokes the release of the Ca2+ accumulated within the endoplasmic reticulum. Analyzing the kinetics of channel opening by the methods of rapid mixing, rapid filtration or flash photolysis of caged InsP3 has revealed that InsP3 opens the channel within a very short time, probably less than 30 msec. The InsP3 receptor-channel is autoregenerative. With the sustained stimulation of a Ca2+ influx the release of Ca2+ leads to an augmentation of [Ca2+]i, which is responsible for triggering cellular responses. The complexity of Ca2+ signals produced by stimulated cells has been revealed by studies in which highly effective techniques have been used to detect Ca2+ ions in the cytosol, such as bioluminescent proteins, fluorescent indicators or ionic currents sensitive to Ca2+. It appears that variations in [Ca2+]i induced by stimulation consist of oscillations of which the frequency, but not the amplitude, depends on the concentration of the hormone. Moreover, by summing the images picked up with a video recorder, it has been possible to demonstrate the changes in [Ca2+]i at the subcellular level and the waves of Ca2+ in stimulated cells.

Effects of chemical modification of Anabaena flavodoxin and ferredoxin-NADP+ reductase on the kinetics of interprotein electron transfer reactions.

The influence of chemical modification of arginine residues (using phenylglyoxal) in ferredoxin-NADP+ reductase (FNR), and of carboxyl groups (using glycine ethyl ester) in flavodoxin (Fld), on the kinetics of electron transfer between FNR and Fld, and between ferredoxin (Fd) and FNR, was examined using laser flash photolysis methods. All proteins were obtained from the cyanobacterium Anabaena PCC7119. Reduction by laser-generated 5-deazariboflavin semiquinone of the FAD moiety of phenylglyoxal-modified FNR occurred with a second-order rate constant 2.5-fold smaller than that obtained for reduction of native FNR, indicating either a small degree of steric hindrance of the cofactor, or a decrease in its redox potential, upon chemical modification. In contrast, no changes were found in the kinetics of reduction of the FMN cofactor of Fld modified by glycine ethyl ester as compared with the native protein. The observed rate constants for reoxidation of Fdred (reduced Fd) by FNRox (oxidized FNR) were dramatically decreased (approximately 100-fold) when phenylglyoxal-modified FNR was used. In contrast to the reaction involving the native proteins, no ionic strength effects on kobs values were found. These results, and those obtained upon varying the protein concentration, indicate that the rate constant for complex formation and the attractive electrostatic interaction between the two proteins were greatly diminished by chemical modification of arginine residues of FNR. When phenylglyoxal-modified FNRsq (FNR semiquinone) was used to reduce Fldox (oxidized Fld), similar inhibitory effects were observed. In this case, the limiting first-order rate constant for Fldsq (Fld semiquinone) formation via intracomplex electron transfer from FNRsq was approximately 12-fold smaller than that obtained for the native FNR (600 s-1 vs 7000 s-1). Again, ionic strength effects were diminished. The glycine-ethyl-ester-modified Fld yielded a limiting first-order rate constant for intracomplex electron transfer from FNRsq to Fldox which was approximately 7-fold smaller (1000 s-1) than that obtained with native Fld, and ionic strength effects were again diminished. These results indicate that complex formation can still occur between modified FNR and native Fld, and between native FNR and modified Fld, but that the geometry of these complexes is altered so as to decrease the effectiveness of interprotein electron transfer. The results are discussed in terms of the specific structural features of the proteins involved.

Laser flash photolysis studies of electron transfer mechanisms in cytochromes: An aromatic residue at position 82 is not required for cytochrome c reduction by flavin semiquinones or electron transfer from cytochrome c to cytochrome oxidase

The influence of an aromatic side chain at position 82 of yeast iso-1-cytochrome c on the kinetics of its electron transfer reactions has been investigated using laser flash photolysis methods to compare a series of site-specific mutant cytochromes in their reduction by free flavin semiquinone and in electron transfer from reduced cytochrome to bovine cytochrome c oxidase. Although small (approximately 10%) but significant differences are observed between some of the mutants (S82, Y82, I82) and wild-type (F82) or G82 cytochrome in the second-order rate constant for reduction by lumiflavin semiquinone, these do not correlate with side-chain aromaticity. In the reaction between the ferrocytochromes and cytochrome c oxidase, significantly larger deviations from exponentiality are found for those mutants having aliphatic residues at position 82 than for wild type or Y82. We interpret the nonexponential behavior in terms of multiple orientations of the cytochromes within the oxidase binding site; the extent to which this occurs is apparently influenced by the character of the residue at position 82. However, a comparison of the average rate constants for electron transfer to cytochrome oxidase for the various mutants reveals that all are closely comparable to WT, except for I82 which is significantly slower (approximately threefold). These results, combined with those obtained previously from steady-state kinetic and thermodynamic measurements, suggest that the observed differences among the mutants are due to alterations in the mode of binding of the cytochrome to the oxidase, rather than to a specific requirement for the presence of an aromatic group at position 82.

Photoinduced charge transfer in rhodamine B/anthracene mixed Langmuir-Blodgett multilayers

The photoexcited transient states of octadecylrhodamine B (R) and 2-(9-anthroyloxy) stearic acid (A) mixed in a stearic acid matrix have been studied by flash photolysis and picosecond fluorescence methods in Langmuir-Blodgett films. The two dominant excitation energy relaxation processes for the R monomer are an electron transfer from A to the excited singlet state of R and an energy transfer to the non-fluorescent R dimer. The excited R dimer relaxes via its triplet state. A charge separation process takes place in the triplet state leading to a radical pair.

Photochemical reactions of dinuclear iron complex Cp2Fe2(CO)4 with tributyltin hydride

The photochemical reactions of Cp 2 Fe 2 (CO) 4 with HSnBu 3 in hexane solutions have been studied by employing continuous or flash photolysis methods.

Study of photocycle and spectral properties of bacteriorhodopsin in Langmuir-Blodgett films

Langmuir-Blodgett (LB) films of bacteriorhodopsin (BR) in soya-phosphatidylcholine (PC) matrix were prepared on quartz plates and the photocycle and spectral properties of the films were studied. The transient absorption spectrum of BR LB film was measured by the nanosecond laser flash photolysis method in a wavelength range from 380 to 650 nm. The absorption maxima were at 540 and 415 nm for the L and M states respectively. In water the rate of decay of the L state was 2.3 × 10 4 s −1 and the rates of formation and decay of the M state were 2.3 × 10 4 s −1 and 3.0 × 10 2 s −1 respectively. The photocycle reaction was not observed in nitrogen-dried BR LB films, but when the films were put back in water, even after being stored for 8 months in dry conditions, the phenomenon could be observed. The energy transfer from trytophan to BR retinal takes place in both dry and wet LB films. The films were relatively stable to changing physical conditions as well as to laser excitation and no changes were observed following repeated photocycles. Molecular surface potentials of BR-soya-PC and soya-PC Langmuir films as a function of mean molecular area decreased towards zero with increasing surface pressure.

Kinetic studies on CO binding to reconstituted myoglobins with four synthetic hemes; structural control in ligand binding to myoglobin

Examination was made of CO binding reactions to four kinds of modified sperm whale myoglobin (Mb), whose heme was reconstituted by iron complexes of synthetic porphyrins such as porphine (Por), meso-tetramethylporphyrin (TMeP), meso-tetraethylporphyrin (TEtP) and meso-tetra(n-propyl)porphyrin (TnPrP), using flash photolysis and stopped-flow methods. The CO association rate was found to be 5- to 20-times and dissociation rate 10- to 36-times accelerated by replacement with synthetic hemes. These features could be explained based on characteristic structures of modified Mbs indicated by X-ray crystallography. The side chain of Arg-45 protruded from the heme vicinity into the solvent region and heme was tilted by interactions of meso-alkyl side chains with surrounding peptides, resulting in the formation of widely opened channels and pockets for ligand passage. These structural features indicate the CO ligand to more easily enter or exit from heme pockets of reconstituted myoglobins, compared to native Mb.

Pulse Radiolysis-Laser Flash Photolysis Studies of Diphenyl Sulfide in Liquid Halocarbons

Abstract The combined pulse radiolysis-laser flash photolysis method has established that the excited state of diphenyl sulfide/chlorine atom complexes readily undergoes intermolecular hydrogen abstraction, while the excited state of diphenyl sulfide/bromine complexes is de-excited without any reactions.

Photochemistry of the uranyl ion with alkenes in acetone—water mixtures

Quenching reactions of the excited uranyl ion with various alkenes (1-heptene, 1-hexene, 1-octene, cycloheptene, cyclohexene, 1,4-cyclohexadiene, 2,4-hexadiene and 1,3-cyclohexadiene) were studied in aqueous 50% acetone mixtures. The quenching constants k q of various alkenes were determined by measuring the emission intensity and by the laser flash photolysis method. A plot of log k q vs. the free-enthalpy change Δ G 2 for the photoinduced electron transfer showed a linear relationship, but the slope (1.0 eV −1) was much smaller than that predicted from the Rehm—Weller correlation. This indicates that direct electron transfer from alkene to excited uranyl ion was not the rate-determining step. These quenching reactions resulted in the formation of uranium(IV), except for the reactions with alkenes of high vertical ionization potentials. The quantum yields for the formation of uranium(IV) were measured. A plot of the reciprocal quantum yield for the reaction with 1,4-cyclohexadiene vs. its reciprocal concentration gave a straight line.

Photolysis of pyrene and chloropyrene in the presence of triethylamine in acetonitrile: dehalogenation assisted by potassium cyanide

Kinetics and transient absorption spectra of pyrene– and 1-chloropyrene–triethylamine (TEA) systems, in the presence and absence of cyanide ions, have been studied by a nanosecond time-resolved laser flash photolysis method in dry acetonitrile at room temperature. Schemes for chloropyrene–triethylamine– and chloropyrene–triethylamine–CN– systems have been developed and individual rate constants determined either by direct measurements or by use of computer analysis of the experimental decay curves. The reaction results in the dehalogenation of chloropyrene to yield pyrene as the reaction product, but high quantum yields (0.2–0.4) are obtained only in the presence of cyanide ions. The hydrogen atoms which replace the chlorines originate from the solvent. This was determined by deuterium labelling. The reaction proceeds via an exciplex to radical ions, which recombine to form ion pairs. Cyanide ions attract chlorohydropyrene radicals, which are mainly formed in the reaction between chloropyrene anion radicals and the solvent. The cyanohydropyrenyl radicals formed are in equilibrium with the relatively stable cyanopyrene anion radicals and, by cleavage of the cyano radicals, yield the reaction product.

Chlorophyll photosensitized electron transfer reactions in lipid vesicles: enhancement in yield of vectorial electron transfer across the bilayer from reduced cytochrome c to oxidized ferredoxin by addition of valinomycin plus potassium ion.

Chlorophyll photosensitized electron transfer across a vesicle bilayer from reduced cytochrome c in the inner compartment to oxidized ferredoxin in the outer compartment, using propylene diquat as a mediator, has been investigated using both steady-state and laser flash photolysis methods. One of the factors limiting the quantum yield is the transmembrane potential, which is formed during sample preparation and is increased by the electron transfer process across the membrane bilayer. This limitation can be diminished by the incorporation of valinomycin into the bilayer in the presence of potassium ion. The overall quantum yield can be approximately doubled (up to a total of 22% based on the chlorophyll triplet which is quenched, and 2.8% based on the absorbed quanta) by valinomycin addition. Another quantum yield limitation arises from the accumulation of oxidized cytochrome c in the inner aqueous compartment, which is formed as a consequence of the transbilayer electron transport process and can quench triplet chlorophyll on the inner side of the vesicle. The chlorophyll cation radical generated in this way can participate in the electron exchange equilibrium between chlorophyll molecules located within the bilayer, and thus inhibit electron flow from inside to outside. This acts to limit the extent of cytochrome c oxidation to less than or equal to 50% of the original amount.

Kinetics of ring‐opening radical polymerization of vinyloxiranes

AbstractRing‐opening radical polymerization has been followed by the flash photolysis method. As an initiator radical, the arylthiyl radical (ArS˙) was used; the addition reaction rate constants of ArS˙ to vinyl monomer containing epoxy‐ring such as vinyloxiranes have been determined at first. The rate constant of ring opening reaction of the adduct radical changing to ArSCH2CH  CROCH2˙ were determined in the form of the relative values, which were converted to the absolute values. The ring‐opening reaction rate constants were ca. 106 s−1, which indicates that the ring‐opening rates are faster than usual radical propagation rates. The rate constants for vinyl monomer with 5‐member ring (1,4‐epoxy‐1,4‐dihydronaphthalene) were similarly evaluated.

Photochemistry of the μ-hydroxo-μ-peroxo- bis[triethylenetetraamine)cobalt(III)] complex in basic aqueous solutions

Photochemical reaction of the title complex [(trien)Co(μ-OH,O 2)Co(trien) 3+ ( 1) was studied in basic, aqueous solution. The photo-induced cobalt(II)-trien complexes, [Co(trien)(H 2O) 2] 2+ and [Co(OH)(trien)(H 2O)] +, react with dioxygen in oxygenated basic, aqueous solution to give the intermediates, [(OH)(trien)Co-O 2-Co(trien)(H 2O)] 3+ and [(OH)(trien)Co-O 2-Co(trien)(OH)] 2+, which are transformed reversibly into 1. The transient absorption spectra of the monobridged μ-peroxo complexes, which have an absorption maximum near 313 nm, were measured using a flash photolysis method. The rate constants of the reaction of cobalt(II)-trien complexes with dioxygen were measured as a function of pH. A small amount of [Co(trien)(H 2O) 2] 3+ ( 2) was detected on prolonged irradiation in both the presence and absence of dioxygen. The photochemical reaction mechanisms are discussed, and the kinetic data of the reaction of cobalt(II)-trien complexes with dioxygen are compared with those obtained by a stopped-flow method.

Flash photolysis study of initiation step in radical polymerization of Si-containing vinyl monomers: reactivities to arylthiyl radicals

The initiation step in radical polymerization of Si-containing vinyl monomers with arylthiyl radicals (ArS.) has been studied by the flash photolysis method. Rate constants for reversible addition reactions of ArS. with various Si-containing vinyl monomers have been determined by the selective radical scavenging method. For each thiyl radical, the reactivities for CH2 CHX increased in the order:X= C(CH3)3 (CH2)3 CH2Si(OCH3)3⩽ CH2Si(CH3)3∼ Si(OCH3)3∼ Si(CH3)3 C6HSi(CH3)3−p∼ C6H4Si(CH3)3−p The reactivity of the C C was increased by the substitution of the Si atom at vinyl and allyl positions. The polar and resonance factors of Si-containing monomers were evaluated from the kinetic data, which were compared with the Alfrey-PriceQ-e scheme obtained from the copolymerization data.

Synthesis, electrochemistry, and kinetic investigations of low-spin ferrous bis(difluoroboryl)bis(dioximate) complexes

The synthesis of new trans-Fe(dioxBF 2 ) 2 (CH 3 CN) 2 complexes (diox=dimethylglyoximate, naphthoquinone dioximate, benzoquinone dioximate) complexes and their conversion to a variety of Fe(dioxBF 2 ) 2 TL complexes (T or L=acetonitrile, 1-methylimidazole, pyridine, tributyl phosphite, tributylphosphine, tert-butyl isocyanide, tosylmethyl ioscyanide, carbon monoxide) is described. Kinetic and equilibrium data for the CH 3 CN derivatives obtained by spectrophotometric and flash-photolysis methods in acetonitrile solution and in toluene are compared with data for other FeN 4 systems

Microtubule assembly and oscillations induced by flash photolysis of caged-GTP

Microtubule assembly and oscillations have been induced using the rapid liberation of GTP by UV flash photolysis of caged-GTP and monitored by time-resolved X-ray scattering. The flash photolysis method of achieving assembly conditions is much faster than the temperature jump method used earlier (msec vs. s range). However, the structural transitions and their rates are similar to those described previously. This means that the rates of the transitions in microtubule assembly observed before are determined by the protein itself, and not by the rate at which assembly conditions are induced. The advantages and limitations of using the photolysis of caged-GTP in microtubule assembly studies are compared with temperature jump methods. Caged-GTP itself reduces the rate of microtubule assembly and oscillations at mM concentrations, consistent with a weak interaction between the nucleotide analogue and the protein. X-rays are capable of slowly liberating GTP and other breakdown products from caged-GTP, even in the absence of UV flash photolysis, thus causing an apparent "X-ray-induced" microtubule assembly. This effect depends on the X-ray dose but is independent of the caged-GTP concentrations used here (mM range), suggesting that the breakdown of caged-GTP is caused not by the direct absorption of X-rays by the compound but by another intermediate reaction such as the generation of radicals by the X-rays.

QUINONE SENSITIZED ELECTRON TRANSFER PHOTOOXIDATION OF NUCLEIC ACIDS: CHEMISTRY OF THYMINE AND THYMIDINE RADICAL CATIONS IN AQUEOUS SOLUTION*

The 2-methyl-1,4-naphthoquinone (MQ) sensitized photooxidation of nucleic acid derivatives has been studied by laser flash photolysis and steady state methods. Thymine and thymidine, as well as other DNA model compounds, quench triplet MQ by electron transfer to give MQ radical anions and pyrimidine or purine radical cations. Although the pyrimidine radical cations cannot be directly observed by flash photolysis, the addition of N,N,N',N'-tetramethyl-1,4-phenylenediamine (TMPD) results in the formation of the TMPD radical cation via scavenging of the pyrimidine radical cation. The photooxidation products for thymine and thymidine are shown to result from subsequent chemical reactions of the radical cations in oxygenated aqueous solution. The quantum yield for substrate loss at limiting substrate concentrations is 0.38 for thymine and 0.66 for thymidine. The chemistry of the radical cations involves hydration by water leading to C(6)-OH adduct radicals of the pyrimidine and deprotonation from the N(1) position in thymine and the C(5) methyl group for thymidine. Superoxide ions produced via quenching of the quinone radical anion with oxygen appear to be involved in the formation of thymine and thymidine hydroperoxides and in the reaction with N(1)-thyminyl radicals to regenerate thymine. The effects of pH were examined in the range pH 5-8 in both the presence and absence of superoxide dismutase. Initial C(6)-OH thymine adducts are suggested to dehydrate to give N(1)-thyminyl radicals.

Studies on photochemically produced metal complexes with coordinated free radicals

In cobalt(III) amine complexes containing amino acids, photolysis in the chargetransfer bands leads to the formation of alkyl cobalt(III) complexes. Steady and flash photolysis of complexes [Co(LL)2X]2+ and [Co(LL)2Y2]+ (LL = ethylene diamine, polypyridyl or propylenediamine, X = amino acid and Y = nitro ligand) were carried out. Steady irradiation of [Co(bpy)2(gly)]2+ ion (bpy = bipyridine, gly = glycine) in aqueous medium (pH 1–7) produces the six-coordinated complex [Co(bpy)2(CH2NH2)]2+. Flash photolysis of the complex under identical conditions gives an alkyl complex. The alkyl complex formed in the case of [Co(bpy)2(gly)]2+ ion is stable and could be isolated; the corresponding alkyl complex formed from [Co(bpy)(en)(gly)]2+ ion leads to the labilisation of the ethylenediamine ligand in acidic solutions. Photolysis of the nitro complexes of the type [Co(en)2(NO2)2]+ ion in acetonitrile, methanol and ethanol produces aπ-bonded transient nitro complex which reacts with molecular oxygen to give the mononuclear superoxo, dinuclear superoxo and dinuclear peroxo complexes in sequence. The formation of these products from the free radical coordinated cobalt(II) complex has been followed by the flash photolysis method and the rate constants have been determined. The details of the mechanisms are discussed.

A new dual‐parameter for reactivities of vinyl monomers toward free‐radicals

AbstractA new dual‐parameter of vinyl monomers for the reactivities toward free‐radicals has been evaluated on the basis of kinetic data obtained by the flash photolysis method. As a model reaction system, the reversible addition reactions of the substituted phenylthiyl radicals to vinyl monomers are selected. The absolute rate constants and the relative equilibrium constants for these reactions have been determined. The Hammett reaction constants (ρ) for the addition reaction rates were evaluated by the variation of the substituents at the para‐position of the arylthiyl radicals. From the values, the polar parameters (P‐ values) have been evaluated. The P values of various vinyl monomers correlate with the e values of the Q‐e scheme proposed by Alfrey and Price. The resonance stabilization terms of the propagating radicals were evaluated from the equilibrium constants (K) which correlate with the Q values. The K‐P dual‐parameter can be used in order to predict the reactivities of vinyl monomers for the primary radicals (initiation step). The monomer reactivities from the copolymerization data are also correlated with the dual parameter. There are some advantages in the new dual‐parameter compared with the Q‐e scheme; the separation of the resonance term from the polar term is complete and the steric factor is not included in the polar term.

ChemInform Abstract: Structure, Spectroscopy and Kinetics of the Methylene Amidogen (H2CN) Radical

The physical and chemical properties of methylene amidogen have been reviewed. In general only limited quantatative information is available about the radical and furhher work, both theoretical and experimental, is required. Theoretical studies have met with success but there is much to be done concerning the electron distribution in the ground state of the radical and the structure and energetics of the excited states. While the ground-state geometry seems well defined theoretically, there is still no experimental confirmation of the calculations. A high resolution IR study shoudd provide this confrrmation and may also lead to the development of more sensitive detection techniques such as IR laser absorption spectroscopy and laser magnetic resonance. Should there techniques prove feasible, then coupling of them to the discharge-flow and flash-photolysis methods will allow us to greatly increase our knowledge of the reactivity of H2CN. We have also reviewed several laboratory and atmospheric systems in which H2CN plays a promintnt role. A better understanging of the kinetics of H2CN will lead to improved models of theee complex systems and additional spectroscopic data makes more feasible the in situ detection of thss important chemical intermediate.