A PULSE RADIOLYSIS STUDY OF THE ONE-ELECTRON OXIDATION AND REDUCTION OF 5, 10-METHENYLTETRAHYDROFOLATE

Abstract— The one-electron reduction and oxidation of 5,10-methenyltetrahydrofolate has been studied in aqueous solution in the acidity range H₀= -1 to pH = 7 using the reducing species CO^-₂ and (CH₃)₂-COH and oxidising species Br^-₂, and H₂SeO+₃. The spectral and other properties of the radicals so formed were found to be indcpendent of the reductant/oxidant used. Two protolytic forms of both the oxidised and reduced radicals were observed with approximate p K , values of 0.5 ± 0.3 being determined. Both the bridged form (5.10-methenyltetrahydrofolate) and the unbridged form (5- formyltetrahydrofolate) were found to be easily oxidised, whereas only the former could be reduced.     

PROTONATION AND DEPROTONATION EQUILIBRIA OF LUMICHROME

Abstract— In order to investigate the possibility of the tautomerization of alloxazine to isolloxazine in its ground state, the parameters affecting the redistribution of charges in the lumichrome molecule were studied. The absorption and emission spectra of lumichrome as a function of pH in the range H₀ = - 6 to pH = 12 were recorded. At extreme pH conditions the spectra of lumichrome are similar to those of the isoalloxazine system. At high acid concentration ( H ₀ < - 3.0) the absorption spectrum of lumichrome protonated at N₁₀, is practically identical to that of lumiflavin. The fluorescence quantum yield of the two cations is negligible at room temperature.
At pH = 10.5 lumichrome is deprotonated at positions N₁ or N₃, The two monoanions have different excitation spectra. Except for slight differences in the extinction coefficients, the absorption of the anion deprotonated at N₃ is very similar to the lumichrome spectrum. The absorption spectra of the N₁ monoanion and of the di-anion are similar to the spectrum of lumiflavin except for a blue shift of about 20 nm. Furthermore, the emission spectrum of the N₁ -monoanion is identical to that of the isoalloxazine system. These results indicate that the charge distribution in the lumichrome molecule depends on the protonation and deprotonation of the nitrogen atoms at positions 10 and l. Both processes cause a redistribution of charges so that an isoalloxazine ring system is formed.     

ROSEOFLAVIN AS A BLUE LIGHT RECEPTOR ANALOG: SPECTROSCOPIC CHARACTERIZATION

Abstract— The blue light absorption band of roseoflavin is polarized along the axis roughly connecting N₃-C₈ positions. A weak, second π→π* transition with a polarization angle of ca. 25° is hidden under the short wavelength side of the blue absorption band. The excited state of roseoflavin is somewhat more basic than the ground state, by a 1.5 p K _a unit. The fluorescence quantum yield and lifetime of roseoflavin are substantially lower than those of other flavins, thus making it kinetically less efficient as a blue light photoreceptor.     

THE USE OF NEUTRAL RED TO MONITOR THE SURFACE POTENTIAL OF THE PURPLE MEMBRANE

Abstract— The binding of neutral red to purple membrane has been studied. The intrinsic p K _a and the apparent p K _a, of bound neutral red were determined by titration and by measuring the binding ratio of neutral red to purple membrane as a function of pH. The surface potential of purple membrane was inferred from the difference between these two p K _as. The H⁺/M412 ratio at different ionic strengths was also measured and compared with the surface potential. The results show that the H⁺/M412 decreased as the surface potential increased due to decreased salt concentrations. However, this correlation holds only for KCl concentrations higher than 30 m M . At lower salt concentrations, the change in surface potential is always less than the variation in the H⁺/M412 ratio.     

A FLASH PHOTOLYSIS STUDY OF 5-METHOXYINDOLE

Abstract— The microsecond flash photolysis of 5-methoxyindole in aqueous solutions has been studied at γ_exc≥ 290 nm. Transients identified in this time realm in neutral solutions are: e_aq^-, the 5-methoxyindole radical cation (γ_max≅ 440 nm), the neutral transient with γ_max≅ 530 nm) and an unidentified oxygen sensitive transient with γ_max≅ 435 nm. Radical cations and e^-_aq are shown to be produced in equal amounts consistent with a photoionization process as the only source of both transients. H⁺ quenching of fluorescence and radical cation production gives equivalent Stern-Volmer constants indicating that photoionization occurs from the fluorescent state. The unidentified oxygen sensitive transient exhibits a pK _a of2–2.5 and is quenched at lower pH values indicating that it also has a fluorescent state precursor.     

PRIMARY PROCESSES IN THE LASER FLASH PHOTOLYSIS AND PULSE RADIOLYSIS OF l-EPHEDRINE

Abstract— Transient absorption spectra produced by laser flash-photolysis of an aqueous solution of ephedrine have been measured under a variety of conditions. Ephedrine was found to photoionise via a biphotonic process. The apparent yield of photoionisation increases with lowering of pH, a value of 8.8 being found for the p K _a associated with this change. The cation radical absorption spectrum has been determined using the techniques of both pulse radiolysis and laser flash photolysis. The extinction coefficient of the cation at 295 nm was determined to be 1.37 × 10⁴ dm³ mol^-1 cm^-1 and 1.2 × 10⁴ dm³ mol-¹cm-¹ by the two techniques, respectively, at pH 11. It is also shown that the rate constant for electron abstraction by the azide radical to form the ephedrine cation is controlled by protonation of the amine group in the side chain. The ephedrine anion radical spectrum and its extinction coefficient at 305 nm were also determined. The excited states responsible for photoionisation and photodegradation are discussed.     

THE PHOTOPHYSICS OF p-AMINOBENZOIC ACID

Abstract— The lowest-lying allowed UV transition in p -aminobenzoic acid (PABA) is assigned Γ→¹L_a based on quantitative absorption and fluorescence studies, as well as semiempirical PM3 multielec-tron configuration interaction calculations. The oscillator strengths, fluorescence quantum efficiencies and lifetimes are reported for PABA in several polar, nonpolar, protic and aprotic solvents (aerated) at 296 K. Reasonable agreement is found between the observed radiative rate constant and that calculated from the absorption and fluorescence spectra. Shifts in the absorption and fluorescence spectra in aprotic solvents are analyzed in terms of the Onsager reaction field model; results are consistent with an increase in dipole moment of ca 4 D between the relaxed S₀ and S₁, states. No evidence is found for the emission from the amino-twisted form of PABA in all solvents studied although calculations show that the amino-twisted S, state is highly polar, but higher in energy by ca 35 kJ/mol ( in vacuo ). The fluorescence efficiency is excitation wavelength independent in both methylcyclohexane and water. The temperature dependence of the nonradiative rate constant (from S₁) was studied in several solvents. Nonradiative decay may be due to intersystem crossing, which would be fast enough to compete with thermally activated intramolecular NH₂ twisting. The phosphorescence spectrum and lifetime obtained in an EPA glass at 77 K are reported, and the triplet energy of PABA is estimated.     

TRANSIENT PHOTOCHEMISTRY OF SAFRANIN-O

Abstract— We have characterized the spectra, acidity constants and decay kinetics of the triplet and semireduced radical species of Safranin-O. Between pH 3.0 and 10.6, there are three triplet species denoted ³DH^{2 +}₂, ³DH⁺ and ³D, the p K _as being 7.5 and 9.2. All three triplet species exhibit first order decay, the rate constant for ³DH⁺ being ca. 5-fold lower than the rate constants of ³DH⁺ and ³D. Ascorbic acid and ethylenediaminetetraacetic acid (EDTA) quench the triplet state under appropriate pH conditions and the pH dependencies of the yield of semireduced dye indicate that ³DH⁺ is more reactive than ³DH⁺ or ³D. With EDTA as the reducing agent, there is the additional requirement that at least one of the amino nitrogens be deprotonated to obtain a significant yield of semireduced dye. In these reactions, ascorbic acid is oxidized reversibly, but EDTA is oxidized irreversibly, so that with the latter reducing agent photolysis causes buildup of the leucodye, which on subsequent photolysis can reduce triplet state dye. With ascorbic acid as the reducing agent, the regeneration of the ground state dye is reversible, the decay of the semireduced radical being second order. In general, the transient photochemistry of Safranin-O resembles that of Thionine, the major difference being that the lifetimes of ³DH^{2 +}₂ and ³DH⁺ are much longer for Safranin-O than for Thionine.     

SPECTROSCOPY OF POLYENES–III. ABSORPTION AND EMISSION SPECTRAL INVESTIGATION OF POLYENE SCHIFF BASES AND PROTONATED SCHIFF BASES RELATED TO VISUAL PIGMENTS

Abstract— A number of n -butylamine Schiff bases of polyenals related to retinals as homologues and analogues, and their protonated forms, have been studied for absorption and emission spectral properties. The polyene Schiff bases exhibit the same general features in their absorption spectra as those of the parallel polyenals except that the ^lB_u←¹A_g and π*← n singlet transitions are at substantially higher energy in the Schiff bases (the shift being larger for the π *← n transition). The Schiff bases with short polyene chainlength ( n = 2, 3 where n is the number of double bonds including C=N) do not fluoresce or phosphoresce in 3-methylpentane in the temperature range 298–77 K. The Schiff bases with intermediate chainlength ( n = 4, 5) show fluorescence at 77 K with intensity strongly dependent on the nature of solvent. The Schiff bases with relatively long chainlength ( n = 5–7) show strong or moderately strong fluorescence at 77 K and very weak fluorescence at 298 K ( n = 7) with intrinsic radiative lifetimes much longer than those estimated from the oscillator strength of the low-energy, strong absorption band (¹B_u←¹ A_g ). A discussion on the possible state order and nature of the fluorescing state of the various polyene Schiff base systems is presented.     

DETERMINATION OF THE pk VALUES OF THE LUMIFLAVIN TRIPLET STATE BY FLASH PHOTOLYSIS

Abstract— The triplet-triplet absorption spectra in aqueous solution of the acid (³LfH₂⁺), the neutral (³LfH) and the basic (³Lf^-) forms of lumifiavin (6,7,9-trimethylisoalloxazine) were measured by flash photolysis. The p K _a values of the corresponding protolytic equilibria of the lumifiavin triplet were found to be 4.4₅±0.1 and 9.8±0.15.     

BIOLUMINESCENCE OF THE BRITTLE STAR OPHIOPSILA CALIFORNICA

The light-emitting principle of the brittle star Ophiopsila californica has been isolated and purified. It was found to be a green-fluorescent photoprotein (molecular weight 45000) which emits green light (λ_max 500 nm) when H₂O₂ is added, independently of the presence or absence of O₂. The green fluorescence (emission maximum 500 nm, excitation maximum 440 nm) spectrally coincided with the H₂O₂-triggered luminescence, indicating that the green fluorescent chromophore is the light-emitter of the photoprotein luminescence.     

CHEMILUMINESCENCE IN THE PROCESS OF OXIDATIVE RING-OPENING OF PURPUROGALLINQUINONES

Abstract— The oxidation of purpurogalline (PPG) by alkaline solution of H₂O₂ pH 9–11 at 298°K is accompanied by chemiluminescence (CL) in the spectral range 400–600 nm with the maximum at 500 nm and quantum yield about 10^-6. The optimal concentrations of reactants with respect to maximal intensity are: 2 × 10^-4 M PPG, 10^-2 M NaOH, 1 M H₂O₂. Activation energy calculated from the maximum intensity of CL is 8.1×0.4 kcal/mole. Light emission occurs only when OH-groups of the phenolic ring of PPG undergo oxidation and the blue anion of o -PPG-quinone is formed. The rate that determines step in the reaction associated with luminescence is the nucleophilic attack of OOH^- ion on the blue anion of o -PPG-quinone. In this exergonic step (-ΔH = 63 to 230kcal/mole) the o - and/or p -quinone ring is opened and carbonyl derivatives of α-tropolone are produced. They display fluorescence in the region 400–600 nm. The fluorescence spectrum of the reaction mixture after oxidation of PPG is very close to that of CL. It is likely that carbonyl derivatives of α-tropolone are emitters of CL.     

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.     

LUMINESCENCE STUDIES OF QUINIZARIN AND DAUNORUBICIN

Abstract— The absorption and emission spectra of quinizarin (1,4-dihydroxy-anthraquinone) have been investigated in hydrocarbon and alcoholic solvents. Fluorescence spectra in 3 different Shpolskii matrices were recorded at 14 K. Vibrational analyses of these spectra revealed the presence of 3, 8, and 9 sites in octane, heptane, and hexane matrices, respectively. The fluorescence lifetime was found to be 6.5 ns in hexane and EPA. Fluorescence photoselection measurements in EPA (77 K) showed that the first 4 electronic transitions of quinizarin are polarized parallel, parallel, perpendicular, and parallel to the long molecular axis and can be assigned, in order of increasing energy, to ¹B₂, ¹B₂, ¹A₁ and ¹B₂ (ππ*)→¹A₁(C_2v,) transitions, respectively. The fluorescent transition is assigned as ¹B₁ (ππ*)→¹A¹. The absence of phosphorescence is attributed to the intramolecular hydrogen bonding present which displaces the parent anthraquinone n →* states above the ππ* states, thereby rendering the intersystem crossing (S₁-T₁) radiationless pathway inefficient.
Photoselection measurements on daunorubicin, a substituted quinizarin and known anticancer drug, revealed an absorption band polarization pattern identical to that of quinizarin. These results are in part at variance with assumptions used in previous work on the intercalation specificity of daunorubicin with DNA.     

A METHOD FOR THE CORRECTION OF QUANTUM YIELDS OF FLUORESCENCE IN SOLUTIONS

Abstract— The correction factors due to the overlap of absorption and emission spectra have been calculated for various concentrations of fluorescein dye at pH 12 by using the experimentally obtained value of apparent molar absorptivity, ε₂, for the fluorescence radiation. The observed fluorescence intensities for the same-surface detection for a given geometry have been corrected for the secondary emission effect thus calculated, and also for refraction and dilution effects, to give corrected relative quantum yields for the solutions. These have been converted to the absolute values by comparison of the integrated fluorescence quantum spectrum of a dilute solution of fluorescein with that for quinine sulphate in 0.1 N H₂SO₄. The corrected quantum yields compare well with values given in the literature.     

AFFINITY FOR OXYGEN IN PHOTOREDUCTION OF MOLECULAR OXYGEN AND SCAVENGING OF HYDROGEN PEROXIDE IN SPINACH CHLOROPLASTS

Abstract— The apparent K _m for O₂ in the photoreduction of molecular oxygen by spinach class II chloroplasts and photosystem I subchloroplast fragments was determined. In both cases, a value of 2 ∼ 3 μ M O₂ was obtained. The reaction rate constant between O₂ and P-430, the primary electron acceptor of PS I, is estimated to be ∼ 1.5 × 10⁷ M ^-1 s^-1 and the factors affecting the production of superoxide by the photoreduction of O₂ in chloroplasts are discussed. Preliminary evidence is presented indicating the occurrence of an azide-insensitive scavenging system for H₂O₂ in chloroplast stroma.     

LOWEST EXCITED TRIPLET-STATE DISSOCIATION CONSTANTS OF PURINES IN AQUEOUS SOLUTION. SUBSTITUENT EFFECTS*

Abstract— Lowest triplet-state dissociation constants of purine and eight of its derivatives have been determined from the shifts of the 0–0 bands of phosphorescence occurring upon protonation and deprotonation (dissociation), in rigid aqueous solutions at 77°K. Triplet-state dissociation constants (p K _aτ) are found to be significantly different from the corresponding ground–state p K _a-values, differences ranging from 0.6 to 3.8 p K units. In the equilibria between neutral and anion species, purines are more acidic in the triplet state, due to derealization of the negative charge of the triplet-state anion. In the equilibria between cation and neutral species, the basicity of purine, amino, methyl, and benzyl-amino derivatives is increased in the triplet state compared to the ground state. The anomalously low basicity of the halogeno- and methylmercapto-derivatives in their triplet state is attributed to the increased interactions of dir orbitals of halogens and sulfur atoms with π orbitals in the triplet state. In general, substituent effects on the p K _a of purines appear to be parallel in the ground and excited triplet states.     

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.     

INACTIVATION OF PHAGE BY NEAR-ULTRAVIOLET RADIATION AND HYDROGEN PEROXIDE

A series of phage with different genomes (both single-stranded and double-stranded RNA and DNA) was inactivated with hydrogen peroxide (H₂O₂) in various combinations with far-ultraviolet (FUV) and near-ultraviolet (NUV) radiations. In every case but one (a lipid-coated phage), a sublethal H₂O₂ concentration greatly enhanced killing by NUV but not FUV. Moreover, this NUV/H₂O₂ synergism was oxygen independent and there was little if any host cell reactivation upon NUV plus H₂O₂ inactivation. These results suggest that these phage are inactivated by a common mechanism irrespective of nucleic acid composition, but that some phage genomes may be more vulnerable to NUV/H₂O₂ inactivation than others.