FLUORESCENCE OF THYMINE IN AQUEOUS SOLUTION AT 300° K

Abstract— –Fluorescence of thymine in neutral aqueous solution at room temperature has been detected using the multiscaling operation of a multichannel analyzer. The emission maximum (2.96 μm^-1) and 0-0 transition energy (3.37-3.45 μm^-1) are close to those determined at liquid nitrogen temperature in mixed solvents. The quantum efficiency of fluorescence excited at 3.77 μm^-1 is calculated to be 1.04 × 10^-4.
The corrected relative excitation spectrum shows significant differences from the absorption spectrum when both are determined under identical conditions of concentration and spectral bandwidth on the same instrument. The quantum yield of fluorescence decreases about 2-fold as the energy of excitation is increased beyond the 0-0' transition and follows the relation 1/φ°α E _excit..
This behavior is discussed in terms of (a) n π* and ππ* states, (b) emission from a minor tautomer and (c) kinetics of competing deactivation processes.     

THE FLUORESCENCE OF ADENINE. THE EFFECTS OF SOLVENT AND TEMPERATURE ON THE QUANTUM YIELD

Abstract— To investigate the mechanism of the solvent and thermal quenching of fluorescence, the absolute fluorescence quantum yield of adenine has been determined in several alcoholic glasses as a function of the concentration (10^-5-10^-4M) and temperature (77°-298°K). The yield is independent of the concentration but increases with decreasing temperature and increasing bulk cohesion and rigidity of the solvent.
The environmental effects on the yield are attributed to radiationless electronic relaxation processes caused by solute-solvent interaction. Increasing the temperature and decreasing the intra-solvent cohesion cause increases in the interaction and therefore accelerate the relaxation. The rate determining step behaves like a diffusion in the limit of low viscosity.     

THE PHOTOPHYSICS OF MEROCYANINE 540. A COMPARATIVE STUDY IN ETHANOL AND IN LIPOSOMES

Abstract— Absorption and fluorescence emission spectra, fluorescence lifetimes, fluorescence quantum yields, photoisomerization quantum yields and triplet quantum yields were measured for Merocyanine 540 (MC540) in ethanol and in large unilamellar dimyristoyl phosphatidylcholine vesicles. The major differences in the photophysics between the two media are the increase of the fluorescence quantum yield from 0.15 in ethanol to 0.6 in vesicles at 25° C, and the appearance of a second fluorescence decay with a lifetime of 1.87 ns in the latter medium. Upper and lower limits for the photoisomerization quantum yields were determined by combining the data from laser flash photolysis and optoacoustic spectroscopy. The decrease in photoisomerization quantum yield upon incorporation of the dye into the lipid bilayer by a factor 2 suggests that this process competes directly with fluorescence. The temperature dependence of the fluorescence and photoisomerization quantum yields in solution supports this model. In both media MC540 has a very low triplet quantum yield with values 0.002 > (> ø_T > 0.02 in ethanol and 0.01 > ø_T- > 0.09 in liposomes Our data are consistent with the model whereby the dye is incorporated into the lipid bilayer as a monomer with two different orientations and this model is adopted on the basis of the biexponential behaviour of the fluorescence and photoisomer decay.     

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.     

PHOTOCHEMISTRY OF FLAVINS. II. PHOTOPHYSICAL PROPERTIES OF ALLOXAZINES AND ISOALLOXAZINES

Abstract. Pulsed laser photolysis at 347nm has been used to study the transient spectroscopy of alloxazine, lumichrome, lumiflavin, and riboflavin in acidic (pH 2.2) aqueous solution and in ethanol. Intersystem crossing quantum yields (φ_ISC) were determined by a modification of the comparative laser excitation method which utilizes the variation of the triplet yield with intensity in conjunction with a kinetic model for the various photophysical and photochemical processes occurring during the pulse. Fluorescence quantum yields and lifetimes are also reported. Correction for quenching of the excited singlet state by H⁺ ions shows that, in neutral aqueous solution, intersystem crossing for flavins is an efficient process (φ_ISC˜ 0.7) which, in conjunction with fluorescence, accounts for the fate of all absorbed photons. For alloxazine (φ_ISC˜ 0.45) and lumichrome (φ_ISC˜ 0.7) the results are more difficult to interpret owing to interconversion between alloxazine and isoalloxazine structures in the singlet excited state. For all four compounds, the quantum yield of products derived from the singlet excited state is estimated as ˜0.04. There is evidence of biphotonic product formation at high laser energies. In ethanol, where φ_ISC for lumichrome is about twice that of lumiflavin, internal conversion between the excited singlet and ground states appears to be a significant process. Complete triplet-triplet absorption spectra in the region 260–750nm are reported. For lumichrome at pH 2.2 there is spectral evidence for isomeric triplet states which appear to be in equilibrium.     

ANALYSIS OF FLUORESCENCE KINETICS AND ENERGY TRANSFER IN ISOLATED α SUBUNITS OF PHYCOERYTHRIN FROM Nostoc sp.

Abstract— The fluorescence decay profiles, relative quantum yield, and transmission of the phycoerythrin a subunit, isolated from the photosynthetic antenna system of Nostoc sp., were measured using single picosecond laser excitation. The fluorescence decay profiles were found to be intensity independent for the intensity range investigated (4 × 10¹³ and 4 × 10¹⁵ photons-cm-² per pulse). The decay profiles were fitted to a model assuming both chromophores absorb and fluoresce. The inferred total deactivation rates for the two chromophores, in the absence of energy transfer and when the effects of the response time of the streak camera and the finite pulse width are properly included, are 1.0 × 10¹⁰s' and 1.0 × 10⁹ s ¹ for the s and f chromophores. respectively, whereas the transfer rate between the two fluorophorcs is estimated to be 1.0 × 10¹⁰ s⁻¹ giving a s→ f transfer rate on the order of (100 ps)⁻¹. Steady-atate polarization measurements were found to be equal to those calculated using the rate parameters inferred from the kinetic model fit to the fluorescence decays. The apparent decrease in the relative fluorescence quantum yield and increase of the relative transmission with increasing excitation intensity is suggestive of ground state depletion and upper excited state absorption. Evidence suggests that exciton annihilation is absent within isolated α subunits for the intensity range investigated (4 × 10¹³ to 4 × 10¹⁵ photons-cm ² per pulse).     

LUMINESCENCE OF TAUTOMERIC FORMS OF THYMINE MONOANIONS

Abstract— The fluorescence and phosphorescence emission and excitation spectra of monoan-ions of thymine. I-methyl-thymine and 3-methylthymine (in alkaline solutions, 10^-2N NaOH) at 300° and 77°K were measured. It was shown, that at 300°K only 3-HT- tautomeric thymine monoanion fluoresces; at 77°K emissions of both monoanionic species contribute to the observed luminescence spectrum.     

FLUORESCENCE LIFETIME AND QUANTUM YIELD OF PHENYLALANINE AQUEOUS SOLUTIONS. TEMPERATURE AND CONCENTRATION EFFECTS

Abstract— –The influence of concentration and temperature on the fluorescence quantum yield and lifetime (measured by a monophoton technique) of phenylalanine is studied in neutral aqueous solutions (8 × 10^-4-10^-1 M ) over the temperature range 0–70°C. The rate constants for emission, internal conversion and intersystem crossing are evaluated and show that both non radiative processes contribute efficiently to the deactivation of the singlet state. Evidence for excited dimer formation at high concentration is presented. The binding energy of excimers was found to be 0.19 e V.     

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.     

On the photochromism of spiro

The recently synthesized spiro[cyclohexadiene-dihydroacridines] consisting of perpendicularly arranged aroylcyclohexadiene and N -methyl-dihydroacridine moieties were found to have photochromic properties. The reversible photoisomerization from the spiro compound toward a colored merocyanine caused by C–C bond cleavage in the cyclohexadiene was studied by stationary and time-resolved measurements of their optical spectra. The course of the absorption under UV and visible irradiation, respectively, and HPLC analysis of the photoproducts result in the determination of excitation energy-dependent quantum yields for the merocyanine formation and, in reverse, the ring closure, as well as degradation. Whereas the thermal back reaction completely recovers the spiro compound ( k ∼ 6.8 × 10⁻⁴ s⁻¹, T = 22°C), degradation of the merocyanine under irradiation at 480 nm has a probability of about 6%. Picosecond-resolved measurements of the fluorescence and the transient absorption show that photoisomerization occurs via the first excited singlet state within 100 ps depending on the activation barrier.     

COMPUTER SIMULATION OF EXCITON JUMPING STATISTICS AND ENERGY FLOW IN C-PHYCOCYANIN OF ALGAE Agmenellum quadruplicatum IN THE PRESENCE OF TRAPS

Abstract—Energy migration has been studied in C-phycocyanin (C-PC) rods with traps located in the terminal trimer disc, using the Monte Carlo method and the system of differential equations. It has been found that jump time statistics can be described by the function F = C(t/0>)exp(-t/ < t_o>), where C is the constant, t and < t₀ > are, respectively, the exciton jump time and its averaged value for chromophores of the corresponding spectral types (α 84 , β84 or β155). The values < t₀ > were calculated for the cases of C-PC monomers, trimers and higher associates.
The C-PC model, which consists of three hexamers with traps located in the β84 chromophores of the peripheral trimer, was examined. It was found that the total efficiency of excitation capturing, ø_tr, exceeds 90%, provided "local" quantum yield of energy trapping ø₀ > 10%. The ø₀ value influences both the excitation lifetime (τ) and the mean number of excitation jumps (N_iump) before its conversion. For the ø₀ = 100% and 10%, the corresponding lifetimes and numbers of jumps were calculated to be τ= 75 and 155 ps and N_jump= 105 and 222 jumps, respectively.
The dynamics of excitation redistribution along the C-PC rods and the fluorescence kinetics for various ø₀ values were calculated for C-PC chromophores excited by a +, and the correlation between these processes and ø ₀ , was disclosed. The transient processes of excitation redistribution were shown to proceed within a time period t < 30 ps.     

PHOTOPHYSICAL PROPERTIES OF NUCLEIC ACID COMPONENTS—1. THE PYRIMIDINES: THYMINE, URACIL, N, N-DIMETHYL DERIVATIVES AND THYMIDINE

Abstract— Low-temperature (and some room temperature) absorption and emission, fluorescence and phosphorescence, data including quantum yields and lifetimes have been obtained from the title pyrimidine bases as a function of the nature of the solvent environment. Modest vibrational resolution has been observed for the first time in the absorption spectra, particularly for thymine and uracil. The excitation spectra also show structure. The quantum yields of fluorescence (φ_F) and phosphorescence are independent of the excitation wavelength. Thymine, thymidine and uracil have profoundly different photophysical properties in polar-aprotic vs polar-protic solvents. The N, N-dimethyl substitution of thymine and uracil produces photophysical changes comparable to the solvent change for the unsubsti-tuted bases. The species involved in the emission processes is the keto (lactam) form. It is probable that ^1,3(n,π*) state(s) has(have) changed order relative to a lowest ¹(π,π*) state as a consequence of both the solvent change and N, N-dimethyl substitution. The lowest triplet state is assigned as ³(n π*). We propose that an important factor contributing to the previously reported excitation wavelength dependence of φ_F and φ_T1 (φ_isc) for nucleic-acid components is the equilibrium coexistence of H-bonded and non-H-bonded forms each having different photophysical properties. Consideration is given of the impact of the significantly different photophysical properties of nucleic-acid bases as a function of the nature of the solvent upon the photochemical properties.     

FLUORESCENCE QUANTUM YIELDS OF 124-kDa PHYTOCHROME FROM OAT UPON EXCITATION WITHIN DIFFERENT ABSORPTION BANDS

Abstract— A fluorescence quantum yield (emission at650–850 nm) of π= (2.3 ± 0.3)10⁻³ was measured for the red-absorbing form (Pr) of 124-kDa phytochrome from etiolated oat seedlings ( Avena sativa ) upon excitation in the Soret band at Λ^exc= 380 nm. The small difference between this value and the previously determined quantum yield with Λ^exc= 640 nm, π= (3.5 ± 0.4)10⁻³is attributed to a blue-absorbing emitter responsible for the "anomalous" or "blue" emission of the chromoprotein in the region from ca. 400 to 550 nm. The absorption of Pr at 380 nm is consequently somewhat lower than that measured directly from the spectrum. Processes from upper excited states of the Pr phytochromobilin-derived chromophore other than rapid relaxation to the emitting state are not important. A quantum yield of Φ ' 1.2 times 10⁻³ is estimated for the blue fluorescence. The proportion of the blue emitters relative to Pr appears to be relatively high.     

QUANTITATIVE SPECTROFLUORIMETRY-THE FLUORESCENCE QUANTUM YIELD OF QUININE SULFATE

Abstract— By idealizing the design of a typical spectrofluorimeter that measures fluorescence from dilute solutions, we are able to derive an equation that predicts the dependence of the observable amplitude on the concentration of the solute and on several instrumental parameters. The result of the derivation, the fluorescence function, is used together with measurements of rhodamine B fluorescence and colloidal silica scattering to calibrate a spectrofluorimeter.
The fluorescence function and the calibration data are used to determine the absolute fluorescence quantum yield of quinine sulfate in 0–1 N H₂ So₄. The results, Ø₂₅₀=0.582 with excitation at 250 mμ, adn Ø₃₅₀=0.577 with excitation at 350mμ. agree well with the previous evaluation of Melhuish, Ø₃₄₅=0.546.     

AUTOMATIC RECORDING OF ACTION SPECTRA OF PHOTOBIOLOGICAL PROCESSES, SPECTROPHOTOMETRIC ANALYSES, FLUORESCENCE MEASUREMENTS AND RECORDING OF THE FIRST DERIVATIVE OF THE ABSORPTION CURVE IN ONE SIMPLE UNIT

Abstract— A compact, rugged and simply constructed instrument has been designed which measures action spectra of photosynthesis by delivering equal numbers of quanta between 400 and 720 nm to a sample placed upon the cathode of an oxygen sensor. The absorption spectrum is measured delivering equal numbers of quanta over the spectrum to a sample placed directly above a photoreceiver that has been adjusted in wavelength sensitivity to function as a quantum counter. All the details that are recorded by conventional high precision spectro-photometers for samples with relatively broad absorption bands (e.g. chlorophyll, carotenoids, phycobilins and living material such as algal suspensions) are resolved by the instrument. Corrected excitation spectra of fluorescence are obtained with the same instrument. Likewise the first derivative of the absorption curve may be scanned. Such recordings amplify details in the absorption spectrum, and they are particularly useful in analyses of small changes in slope. The functions described above may be operated separately or combined. The instrument has a total weight of about 10 kg, and the dimensions are ca . 60 × 40 × 30 cm. It is constructed for use in field laboratories and on board research vessels, and also for courses in biology where the principles behind photobiological analyses are illustrated. It is possible that a device of this type could be used for investigations of photosynthesis on other planets.     

FLUORESCENT TAUTOMERS AND THE APPARENT PHOTOPHYSICS OF ADENINE AND GUANINE

Abstract— Fluorescence, absorption and fluorescence excitation spectra, and quantum yields of 0.02 mM solutions of adenine, 7-methyladenine (7-MA), guanine and 7-melhylguanine (7-MG) are presented for excitation with240–300 nm light. The solvent is neutral ethylene glycol-water (70:30 v/v) in the temperature range140–165 K. Phosphorescence spectra of adenine and 7-MA at 140 K are also presented. The excitation spectrum of adenine shows vibrational structure, whereas the absorption does not. However, the fluorescence of adenine shows the vibrational structure, as do the absorption, fluorescence and excitation spectra of 7-MA. The results confirm (and reinforce) the notion that luminescence from adenine under these conditions is from the N₇–H tautomer, instead of the more abundant N₉–H form. In a similar fashion, the data from guanine and 7-MG strongly suggest that the luminescence from guanine is also mostly from the N₇–H tautomer.     

LIGHT-INDUCED QUENCHING OF PHOTOSYSTEM II FLUORESCENCE AT 77 K

Abstract— Light-induced quenching of the low temperature fluorescence emission from photosystem II (PS II) at 695 nm ( F ₆₉₅) has been observed in chloroplasts and whole leaves of spinach. Photosystem I (PS I) fluorescence emission at 735 nm ( F ₇₃₅) is quenched to a lesser degree but this quenching is thought to originate from PS II and is manifest in a reduced amount of excitation energy available for spillover to PS I. Differential quenching of these two fluorescence emissions leads to an increase in the F ₇₃₅/ F ₆₈₅ ratio on exposure to light at 77 K. Rewarming the sample from -196°C discharges the thermoluminescence Z-band and much of the original unquenched fluorescence is recovered. The relationship between the thermoluminescence Z-band and the quenching of the low temperature fluorescence emission ( F ₆₉₅) is discussed with respect to the formation of reduced pheophytin in the PS II reaction center at 77 K.     

BINDING, EXCITATION ENERGY TRANSFER AND PHOTOSENSITIZATION IN GRISEOFULVIN-DNA MIXTURES

Abstract— The weak and reversible binding of the antifungal drug, griseofulvin (GF), to calf thymus DNA has been demonstrated by difference spectroscopy and the quenching of the fluorescence of GF by DNA observed. The value of K _n was determined to be 800 M ^-1by fluorescence quenching titration. Adenosine and guanosine also exhibit difference spectra with GF and quench GF fluorescence indicating that they may be the site of both binding and energy transfer. The in vitro photosensitization of DNA by griseofulvin is shown to occur. It is proposed that the clinically observed in vivo photosensitizing action of griseofulvin may result from binding followed by excitation energy transfer and that this may also be important in the antifungal activity of the drug.     

POLARIZED FLUORESCENCE OF 5-METHYLCYTOSINE SPECIES IN SOLUTION AT ROOM TEMPERATURE

Fluorescence yields (π_f,'s) and polarizations ( P ) are measured for aqueous 5-methylcytosine (˜ 0.1 m M ) at 20°C as a function of pH over the range 2–14. Both properties change abruptly and in parallel at pH's corresponding to the known pK_a values. Polarizations were also obtained for the 5-methylcytosine cation, neutral and anion species in ethylene glycol water glass at ˜180K. The weak fluorescence of the neutral and cation species at 20°C was polarized almost as highly as at low temperature. When the fluorescence lifetimes are assumed to be correctly given by the product of calculated radiative lifetimes and quantum yields, the polarizations are found to be consistent with rotational diffusion rates ˜4 times faster than predicted from Stokes-Einstein models for the neutral and anion species. The cation seemed to rotate about two times more slowly than the neutral and anion species. It was also shown that the properties of the three species are such that a plot of 1/P vs apparent π_f in the pH range 2–11 is fortuitously linear.     

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.