LASER FLASH PHOTOLYSIS AND PHOTOINACTIVATION OF SUBTILISIN BPN'

Abstract— Laser flash photolysis of subtilisin BPN'at 265 nm has shown that photoionization of tryptophanyl (Trp) and tyrosinyl (Tyr) residues are the principal initial photochemical reactions. The initial products are the corresponding oxidized radicals. Trp and Tyr, and hydrated electrons (e_aq) which react with the enzyme at: k (e_aq+ subt. BPN') = 2.1 × 10¹⁰ M⁻¹ s⁻¹. The photoionization quantum yield was 0.032 ± 0.005 at 265 nm, which was enhanced 3.5-fold by simultaneous excitation at 265 and 530 nm. The photoionization yields were unchanged by 3 M bromide ion and 8 M urea. which did affect the enzyme fluorescence excited at 265 and 295 nm. A similar lack of correlation between the effects of perturbants on the photionization yields and fluorescence yields was found for subtilisin Carlsherg. The results indicate that the monophotonic and biphotonic ionization of the Trp residues does not involve the thermally-equilibrated. lowest excited singlet state and that singlet energy transfer from Tyr to Trp does not contribute to Trp photoionization. The photoinactivation quantum yield was 0.014 for 265 nm laser excitation. which was not changed by simultaneous 530 nm excitation. The corresponding quantum yield was 0.009 for low intensity 254 nm radiation, indicative of a biphotonic contribution to photoinactivation. The results are explained by postulating that photolysis of Trp-113 leads to disruption of hydrogen bonding to Asn-117 and a shift in the primary chain sequence associated with the aromatic substrate binding sites. The photoionization quantum yields in subtilisin BPN'and subtilisin Carlsberg agree with a model based on the assumption that exposed Trp and Tyr residues contribute independently at intrinsic photoionization efficiencies characteristic of the chromophores.     

KINETICS OF BACTERIAL BIOLUMINESCENCE AND THE FLUORESCENT TRANSIENT

Abstract— The addition of FMNH₂ to Vibrio harveyi luciferase at 2°C in the presence of tetradecanal results in the formation of a highly fluorescent transient species with a spectral distribution indistinguishable from that of the bioluminescence. The bioluminescence reaches maximum intensity in 1.5 s and decays in a complex manner with exponential components of 10^-1s^-1, 7 × 10^-3s^-1, and 7 × 10 ⁴s^-1. The fluorescent transient rises exponentially at 7 × 10^-2s^-3 and decays at 3 × 10^-4s^-1. The slowest bioluminescence component, comprising the bulk of the bioluminescence, decays at twice the rate of the fluorescent transient under all variations of reaction conditions: concentration of reactants, temperature 2–20°C, and aldehyde chain length—decanal, dodecanal and tetradecanal. The activation energy for both the slowest bioluminescence decay and the transient fluorescence decay is 80 kJ-mol^-1. An energy transfer scheme is proposed to explain the results where two distinct chemically energized species utilize the fluorescent transient as emitter for the slower bioluminescences, and for the faster process a fluorophore present in the protein preparation. Kinetic observations suggest that typical preparations of V. harveyi luciferase comprise 15% active protein.     

STUDIES ON SOME INTERMEDIATES AND PRODUCTS OF THE PHOTOREDUCTION OF 9,10-ANTHRAQUINONE *,†

Abstract— The mechanism of the photoreduction of 9,10-anthraquinone (AQ) in alcohol and hexane has been studied by flash photolysis. The fluorescence spectrum of the photoproduct, 9,10-dihydroxy anthracene shows a large shift between hexane and ethanol. The quantum yields of photoreduction for AQ are solvent-dependent, the reaction between the solvent radical and AQ determining the quantum yield.
The absorption spectrum of the 9,10-anthrasemiquinone (AQH.) has a long-wavelength absorption band with peaks at 631 and 678 nm. The second-order decay constants for AQH. were estimated to be 1.3 × 10⁹, 6.7 × 10⁸ and 2.0 × 10⁸ M ^-1 sec^-1 in ethanol, 2-propanol and ethylene glycol, respectively.
A long-wavelength absorption band was observed for 9,10-anthrasemiquinone radical anion, having peaks at 776 and 860 nm; epsi;_max= 1900 at 776 nm. This spectrum is compared with the spectra of 9,10-dihydroxy anthracene mono- and di-anions. The 9,10-anthrasemiquinone radical anion was found to photoreduce quantitatively to 9,10-dihydroxy anthracene mono-anion with a quantum yield of 0.1.     

REACTIONS OF PHOTOCHEMICALLY FORMED TRANSIENTS FROM 2-NITROTOLUENE

Abstract— Kinetic data are reported for the thermal decay of colored transients formed by U.V. irradiation of aqueous solutions of 2-nitrotoluene. The transients display an acid-base equilibrium with a pK value of 3.7. The decay is catalyzed by acids and the following rate constants in liter sec^-l mole^-1 were evaluated for the base form of the transient reacting with an acid at 30.0°C: 3.5 × 10^-3 (H₂O), 2.6×10³ (CH₃COOH), 4.7×10⁴ (⁺NH₃CH₂COOH) and 4.2×10⁵(H+).     

PICOSECOND TIME RESOLVED FLUORESCENCE OF CHLOROPHYLL IN VIVO

Abstract. The published data concerning the fluorescence kinetics of chlorophyll a in various photosynthetic species are reviewed. The effects of singlet-singlet and singlet-triplet annihilation induced by excessively high incident light intensities are discussed and related to the changes produced in the fluorescence lifetimes and quantum yields. We also review the fluorescence lifetimes of Chlorella pyrenoidosa and spinach chloroplast fragments under a variety of experimental conditions; these measurements were performed at single pulse excitation intensities of less than 5 × 10¹³ photons cm^–2 where distortion due to annihilation processes is negligible. Evidence for and against a time dependent rate equation for energy migration will be discussed with reference to the authors' work on in vitro systems.     

FLASH PHOTOLYSIS STUDIES OF OROTIC ACID

Abstract— The triplet state of orotic acid has been studied by flash photolysis. The rate for dimerization has been observed to vary from 2 × 10⁹ M ^-1 sec^-1 at pH 1 where both the triplet and ground state molecules are neutral, to under 10⁸ M^-1 sec^-1 above pH 9 where both the triplet and ground state molecules are doubly ionized. The p K of the triplet state has been measured as 4.6. The rate of oxygen quenching for the triplet is 2–3 × 10⁹ M^-1 sec^-1 while the rate of radiationless decay in solution is 0.73 × 10⁴ sec^-1. The triplet absorption spectra have been measured for the two ionic forms of the triplet.     

CHLORIDE ENHANCEMENT OF QUENCHING OF TRIPLET METHYLENE BLUE BY Fe(II)

Abstract— The influence of chloride ion on the rate of decay of triplet methylene blue in 0.01 M acid in the absence and presence of ferrous ions was investigated by means of laser flash-photolysis monitored by kinetic spectrophotometry. Chloride weakly accelerates decay of ³MBH^2± in aqueous solution in the absence of Fe(II). Quenching of ³MBH²⁺ by Fe(II) is more strongly catalyzed by Cl^- in both water and 50 v/v% aq. CH₃CN. The uncatalyzed quenching constant, k ₅, is of the order of 1 × 10⁶ M ^-1 s^-1 while in 4.8 M aqueous chloride ( μ – 7.2 M ) k ₅= (37.2 ± 1.8) × 10⁶ M ^-1 s^-1. A possible role of chloride is as a bridging species in quenching via electron transfer between ³MBH²⁺ and Fe(II).     

DEUTERIUM-ISOTOPE EFFECT ON THE FLUORESCENCE YIELDS AND LIFETIMES OF INDOLE DERIVATIVES–INCLUDING TRYPTOPHAN AND TRYPTAMINE

Abstract— The fluorescence yields and lifetimes of indole, five of its alkyl detivatives, tryptophan, and tryptamine have been determined in degassed, heavy and light water at room temperature. All of the compounds have radiative lifetimes nearly identical to the parent compound indole, and a comparison of these results with recently reported data on tryptophyl derivatives disclosed a striking uniformity in radiative lifetimes between indole and many amino acids and peptides which contain the indole group as the fluorescence unit. The fluorescence rate k _f in H₂O, was found to be 4.5 × 10⁷ sec^-1. The nonradiative decay rates were found to vary between 5.1 and 46 × 10⁷ sec^-1 and from a study of the deuterium-solvent isotope effect and the deuterium-substituent effect a mechanism for nonradiative deactivation is proposed which includes an isotopically dependent proton transfer and a pathway involving energy loss via the ring carbon hydrogen vibrations. Tryptophan at pH 7 was found to have a unique nonradiative decay scheme not evidenced at a pH 1 or pH 10.     

TRANSIENT PHOTOCHEMISTRY OF NEUTRAL RED

Abstract— Flash photolysis of neutral red between pH 1.3 and pH 11 yields the triplet species ³DH₂⁺²³DH⁺ and ³D. Both ³DH₂⁺² and ³D exhibit first order decay with rate constants of 1.6 ± 0.3 × 10⁴ s^-1 but ³DH⁺ decays within the lifetime of the flash. Over the entire pH range, ascorbic acid quenches the triplet, forming the semireduced radicals DH₃⁺² DH₂⁺ and DH, all of which exhibit second order decay with k = 1.8 ± 0.4 ± 10⁸ M ^-1s^-1 most probably by recombination with semioxidized ascorbic acid. The dependence of the rate of decay of radical neutral red on the identity of reversible reductants supports the back-electron transfer mechanism, as does digital simulation of complex radical disproportionation schemes. In contrast to the efficient reduction of triplet neutral red by ascorbic acid, its reduction by EDTA is quite inefficient.     

PHOTOBLEACHING OF A CYANINE DYE IN SOLUTION AND IN MEMBRANES

Abstract— N,N'-bis(2-ethyl-1,3-dioxolane)-kryptocyanine (EDKC), a lipophilic dye with a delocalized positive charge, photosensitizes cells to visible irradiation. In phosphate-buffered saline (PBS), EDKC absorbs maximally at 700 nm (ε= 1.2 × 10⁵ M⁻¹ cm⁻¹) and in methanol, the absorption maximum is at 706 nm (ε= 2.3 × 10⁵ M⁻¹ cm⁻¹). EDKC partitions from PBS into small unilamellar liposomes prepared from saturated phospholipids and into membranes prepared from red blood cells (RBC) and binds to human serum albumin (HSA). The EDKC fluorescence maximum red shifts from 713 nm in PBS to 720–725 nm in liposomes and RBC membranes and the fluorescence intensity is enhanced by factors of 14–35 compared to PBS (φ= 0.0046). EDKC is thermally unstable in PBS (T_1/2= 2 h at 1.3 × 10⁻⁵ M EDKC), but stable in methanol. In liposomes and RBC membranes, EDKC is 10 times more stable than in PBS, indicating that it is only partially exposed to the aqueous phase. Quenching of EDKC fluorescence in liposomes and RBC membranes by trinitrobenzene sulfonate also indicates that EDKC is not buried within the membranes. Photodecomposition of EDKC was oxygen-dependent and occurred with a low quantum yield (6.4 × 10⁻⁴ in PBS). Singlet oxygen was not detected upon irradiation of EDKC in membranes or with HSA since the self-sensitized oxidation of EDKC occurred at the same rate in D₂O as in H₂O and was not quenched by sodium azide or histidine.     

QUENCHING OF CHLOROPHYLL FLUORESCENCE BY NITROBENZENE

Abstract—Nitrobenzene quenching of chlorophyll fluorescence in ethanol has been investigated. Steady state relative quantum yields have been measured and fluorescence decay rates were determined using both nanosecond photon counting and picosecond pulses from a mode-locked Nd³⁺ glass laser.
The fluorescence decay is described by
1( t )= I ₀ exp (- t/τ−At^1/2 )
the form predicted for decay governed by the kinetics of the continuum model of diffusion controlled reactions. From the parameters of the fluorescence decay, the encounter distance is 5–7 A° the mutual diffusion coefficient is 0.62 × 10^--⁵ cm2s^-1± 12%.
Some of the fluorescence quenching is also attributed to static quenching by a nitrobenzene-chlorophyll, ground-state complex. The equilibrium constant for formation of this ground-state complex was determined to be 4.1 M ^-1. The combined dynamic and static quenching model allows calculation of quantum yields of fluorescence in good agreement with the experimentally determined quantum yields.     

QUENCHING OF EXCITED TRIPLET LUMIFLAVIN BY LUMIFLAVIN RADICALS FORMED IN ITS T-T REACTION

Abstract— Reactions of the triplet state of lumiflavin (³LF) in water adjusted at pH 7.2 were reexamined by means of a Xe-flash photolysis and a laser photolysis. Measurements of the decay of ³LF were made on solutions of LF ranging in the concentration from 4 to 61 times 10^-6 mol/dm³. A one-electron reduced and a one-electron oxidized species of lumiflavin (LF^- and LF⁺) were produced in the first decay stage of ³LF with a high efficiency (0.6 ± 0.1) in a bimolecular triplet-triplet reaction. The product radicals (LFH^- and LF⁺) quench ³LF very efficiently (3 ± 0.8 × 10⁹ mol^-1dm³ s^-1) compared with LF in the ground state (> 2 × 10⁷ dm³ mol^-1).     

LASER PULSE EXCITATION STUDIES OF THE FLUORESCENCE OF CHLOROPLASTS

Abstract. The fluorescence yield, φ, as a function of single picosecond laser pulse intensity was experimentally studied in spinach chloroplasts and for chlorophyll a in ethyl ether solution. The progressive decrease in φ with increasing incident intensity for in vivo chlorophyll was found to be adequately explained within the context of continuum bimolecular kinetics with a singlet-singlet fusion rate constant of γ=5×^-9cm^-3s^-1 at room temperature. We discuss qualitatively how the fluorescence quantum yield depends on the duration and intensity of the incident pulse. The identity of φ vs l (the number of absorbed quanta) curves at the emission maxima of 685 nm and 735 nm for single picosecond pulse mode of excitation is explained within the context of Butler's tripartite model of the fluorescence of chloroplasts at 77 K. Various models relating γ to the singlet exciton diffusion coefficient and the Förster energy transfer rate are used to infer lower bounds to these physical parameters. Predictions and supporting experimental evidence for the tripartite model are discussed.     

THERMAL DENATURATION OF MONOMERIC AND TRIMERIC PHYCOCYANINS STUDIED BY STATIC AND POLARIZED TIME-RESOLVED FLUORESCENCE SPECTROSCOPY

C. Phycocyanin (PC) and allophycocyanin (APC). as well as the α-subunit of PC. have been isolated from the blue-green alga (cyanobacterium). Spirulina platensis . The effects of partial thermal denaturation of PC and of its state of aggregation have been studied by ps time-resolved, polarized fluorescence spectroscopy. All measurements have been performed under low photon fluxes(10¹³ photons/pulse cm²) to minimize singlet-singlet annihilation processes. A complex decay is obtained under most conditions, which can be fitted satisfactorily with a bi-exponential (T₁=70–400 ps, T₂ -1000–3000 ps) for both the isotropic and the polarized part, but with different intensities and time constants for the two decay curves. The data are interpreted in the framework of the model first developed by Teale and Dale [ Biochem. J. 116, 161 (1970)], which divides the spectroscopically different chromophores in (predominantly) sensitizing ( s ) and fluorescing ( f ), ones. If one assumes temperature dependent losses in the energy transfer from the s to the f and between f chromophores. both the biexponential nature of the isotropic fluorescence decay and the polarization data can be rationalized. In the isotropic emission (corresponding to the population of excited states) the short lifetime is related to the sf transfer. the longer one to the "free" decay of the final acceptor( s ) (= f ). The polarized part is dominated by an extremely short decay time. which is related to sf transfer, as well as to resonance transfer between the f -chromophores.     

EXCITED STATES OF ANTHOCYANINS: THE CHALCONE ISOMERS OF MALVIDIN 3,5-DIGLUCOSIDE

Abstract The excited state properties of the chalcone isomers of malvidin 3,5-diglucoside (malvin) in acidic aqueous solution (0 < pH < 4) were investigated using steady-state and time-resolved fluorescence spectroscopy. The two chalcone isomers of malvin were first isolated by high-performance liquid chromatography and then characterized by UV/visible absorption and fluorescence spectroscopy. The results were supported by molecular orbital calculations. The rate constants for photodeprotonation ( k ₁= 1 × 10⁹ s^−l) and protonation ( k ₋₁= 1.3 × 10¹⁰ L mol⁻¹ s^−l) were determined, both from the multiexponential fluorescence decays and the fluorescence intensities measured at the emission wavelengths of the neutral and ionized chalcone forms. The results here obtained are relevant for the understanding of the photoreactivity of anthocyanins in acidic medium.     

SYNTHESES AND PHOTOREDOX PROPERTIES OF POLYELECTROLYTES FUNCTIONALIZED WITH 5-DEAZAFLAVIN

Abstract— Anionic polyelectrolytes functionalized with the 5-deazaflavin group (dFl) were synthesized. The lifetime of the triplet excited dFl in the polyelectrolytes with a 2-mol% dFl content (AdFl-2) was about 10 times longer than that of a low molecular weight analog (AdFl-M). 2-Mercaptoethanol (RSH) reduced the triplet dFl with the rate constant of k _red= 2.01 × 10⁸ M ⁻¹ s⁻¹ for AdFl-M and k _red= 4.4 × 10⁷ M ⁻¹ s⁻¹ for AdFl-2. A zwitterionic viologen (SPV) oxidized the triplet dFl with the rate constant of k _red= 3.69 × 10⁹ M ⁻¹ s⁻¹ for AdFl-M and k _ox= 7.4 × 10⁸ M⁻¹ s⁻¹ for AdFl-2. The smaller rate constants for the polymer system were discussed in terms of the hindering effect of the macromolecular microenvironment. The back electron transfer was shown to be drastically slowed in the AdFl-2-SPV system as a result of the intensive electrostatic effect of the polyelectrolytes. The buildup of the viologen radicals was studied under the steady-state illumination of the three component systems including viologen and RSH. The dFl group was demonstrated to serve as a very efficient photosensitizer in the oxidative cycle in case back electron transfer was retarded. This is the case of the AdFl-2-SPV system which gave the quantum yield of about 0.4 for the SPV⁻ buildup. By comparison, the AdFl-2-MV²+ system resulted in a much slower buildup of MV +radicals.     

FLUORESCENCE OF 5-METHYLCYTOSINE

Abstract— 5-Methylcytosine and 5-methyldeoxycytidylic acid are fluorescent in aqueous solution at room temperature and neutral pH. 5-Methylcytosine, 10^-3M, pH 8.5, 25°C, has a quantum yield of 5 ×10^-4, 5-Methyldeoxycitydylic acid, 10^-4M, pH 7.5, 20°C, has a quantum yield of 8 × 10^-4. Emission maxima are 2.91 and 2.80μ^-1. At pH 14, the quantum yield of 5-methylcytosine is 1.6 × 10^-2; the emission maximum is 2.82μ^-1. At pH I, the quantum yield of both compounds is less than or equal to 10^-4. Both compounds were chromatographically homogeneous, had absorption spectra which agreed with published data, and excitation spectra which agreed closely with absorption spectra.     

pH-DEPENDENCE OF RHODAMINE B SEMIQUINONE DISMUTATION RATE IN AQUEOUS ALCOHOLIC SOLUTION

Abstract— A single transient species is observed in absorption following the flash irradiation of dilute deoxygenated aqueous alcoholic solutions of rhodamine B in hte pH range 5.5–7.0 The second-order decay constant for this semiquinone radical is an approximate linear function of the prevailing hydrogen ion concentration, the transient persisting for a period of from several seconds in neutral solution to several hours at pH = 12.
The slow rate of decay and its pH-dependence are explained in terms of an electron or H-atom transfer between the (unobserved) protonated and (observed) unprotonated semiquinone radical with simultaneous regeneration of hte D^+> and production of the leuco-dye DH:—
D·+ DH·⁺→ D⁺+ DH
Insofar as the reactant concentrations are controlled by the acid dissociation constant K^a of DH^.+ the data are consistent with a value of 5.5±1.0 for pK_a and a pH-independent rate constant K_o of 1.3±0.5×10⁶ 1. Mole^-1 sec^-1 at 20C.     

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.     

A SINGLE PULSE PICOSECOND LASER STUDY OF EXCITON DYNAMICS IN CHLOROPLASTS

Abstract. Using single picosecond laser pulses at 610 nm, the fluorescence yield (φ) of spinach chloroplasts as a function of intensity ( I ) (10¹²-10¹⁶ photons/pulse/cm²) was studied in the range of 21–300 K. The quantum yield decreases with increasing intensity and the φ vs I curves are identical at the emission maxima of 685 and 735 nm. This result is interpreted in terms of singlet exciton-exciton annihilation on the level of the light-harvesting pigments which occurs before energy is transferred to the Photosystem I pigments which emit at 735 nm.
The yield φ is decreased by factors of 12 and 43 at 300 and 21 K, respectively. The shapes of the φ vs I curves are not well accounted for in terms of a model which is based on a Poisson distribution of photon hits in separate photosynthetic units, but can be satisfactorily described using a one-parameter fit and an exciton-exciton annihilation model. The bimolecular annihilation rate constant is found to be γ= (5–15) times 10^-9cm³s^-1 and to exhibit only a minor temperature dependence. Lower bound values of the singlet exciton diffusion coefficient (≥ 10^-3cm²s^-1), diffusion length (≥ 2 times 10^-6cm) and Förster energy transfer rates (≥ 3 ≥ 10¹⁰s^-1) are estimated from γ using the appropriate theoretical relationships.