FLUORESCENCE AND THE LOCATION OF TRYPTOPHAN RESIDUES IN PROTEIN MOLECULES

Abstract— Fluorescence spectra of a number of native and denaturated proteins have been analysed, using spectral band width (ΔΛ), spectral maximum position (Λ_m), fluorescence quenching by external ionic quenchers, lifetime (b), and quantum yield ( q ) and its changes upon denaturation. The results enabled a model of fluorescence properties of tryptophan residues in the proteins to be substantiated by considering the existence of three discrete spectral classes, one buried in nonpolar regions of the protein (Λ_m 330–332 nm, ΔΛ= 48–49nm, q 0.11, τ= 2.1 ns) and two on the surface. One of the latter is completely exposed to water (Λ_m# 350–353 nm, ΔΛ= 59–61 nm, q # 0.2, τ= 5.4 ns); the other is in limited contact with water which is probably immobilized by bonding at the macromolecular surface (Λ_m# 340–342 nm, ΔΛ= 53–55 nm, q # 0.3, = 4.4 ns). Some quantitative predictions from the model, for (a) the fraction of fluorescence that is quenched by ionic quenchers, (b) the mean values of quantum yield, and (c) the mean values of fluorescence lifetime for various proteins, show good concordance with independent experimentally determined values.     

ACID DEPENDENT FLUORESCENCE QUANTUM YIELD AND TETRAMETHYL- 1,2-DIOXETANE CHEMI-ENERGIZED FLUORESCENCE OF ERGOSTATETRAENONE*

Abstract. –In acidified acetonitrile, ergostatetraenone (E) efficiently quenches the tetramethyl-1,2-dioxetane (TMD) chemiluminescence at 410 nm, with the appearance of a new chemiluminescence at ca. 530 nm due to ergostatetraenone fluorescence. Control experiments exclude energization by triplet acetone and the trivial process (emission and reabsorption), establishing singlet-singlet energy transfer between excited singlet acetone (A) and ground state ergostatetraenone as major pathway. From the kinetics the rate constant for singlet-singlet energy transfer ( K ^ss_A,1,) was estimated to be ca. 10^{1 2}S^-1. The chemiluminescence yield (φ^CL) for the TMD/ergostatetraenone system was determined to be ca. 12 × 10^-8. The fluorescence efficiency of ergostatetraenone (φ) in acidified acetonitrile is a function of acid concentration, ranging between 0.06-0.82. The efficiency of singlet acetone production (α) was found to be 500-fold lower than in neutral medium due to a competing acid-catalysed, dark decomposition of TMD. A 60-fold light enhancement has been established for this binary chemiluminescent system.     

CHEMILUMINESCENCE AND FLUORESCENCE OF THE EUROPIUM IONS-ADENINE NUCLEOTIDES SYSTEM AND ITS POSSIBLE BIOLOGICAL SIGNIFICANCE

Abstract— Chemiluminescence of the Eu(II)/Eu(III)-adenine nucleotide-H₂O₂ system and fluorescence of the Eu(III)-adenosine triphosphate system have been investigated. The spectral distribution of the chemiluminescence emission has shown an occurrence of three main bands (Λ=470–480,590–620 and ca. 700 nm). The energy transfer process from the adenosine triphosphate molecules to the Eu(III) ions has been observed in the fluorescence spectrum. The examined chemiluminescence and fluorescence spectra show that these both kinds of emission originate from the ⁵ D _***τ⁷F_*** ( n =1–4) transitions in the Eu(III) ions.     

FLUORESCENCE SPECTRA OF BLOWFLY METAXANTHOPSINS

Abstract— The main visual pigment of blowflies (xanthopsin, Vogt, 1983 Z. Naturforsch. 38c ,329–333) photoconverts into two thermostable metaxanthopsin states M and M'(e.g. Stavenga et al. , 1984 Photochem. Photobiol. 40 ,653–659). The fluorescence spectra of the two photoproducts were studied by microspectrofluorometry in vivo. The emission spectra of M and M'are very similar and peak at 660 nm. The excitation spectra of M and M'have peak wavelengths at Λ_max ' 584 nm and = 568 nm respectively.     

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.     

FLUORESCENCE AND THE STRUCTURE OF PROTEIN—XX. FLUORESCENCE OF 3-AMINOTYROSINE AND OTHER AMINOPHENOLS

Abstract— –The fluorescence of 3-NH₂ tyrosine and of simpler isomeric aminophenols was observed in aqueous solution at room temperature. Marked changes in both emission spectrum and fluorescence efficiency were observed upon change of pH and solvent composition. An explanation for the observed behavior is offered on the basis of two effects. First, peculiarities in the emission spectra were accountable in terms of proton dissociation from the cationic amino group in the excited singlet state so that emission characteristic of the free base was observed even in acidic solutions. Second, the unexpectedly low fluorescence efficiency from the free base was shown to result from quenching by the aqueous solvent. The relevance of these studies for the fluorescence of 3-NH₂ tyrosyl groups in proteins is discussed briefly.     

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.     

EXCITED STATES OF DNA AND ITS COMPONENTS AT ROOM TEMPERATURE—III. SPECTRA, POLARISATION AND QUANTUM YIELDS OF EMISSIONS FROM ApA AND poly rA*

Abstract— Corrected normalised emission spectra from 300 to 480 nm and their polarisation are reported for neutral and poly rA at room temperature. Yields are 4 times (ApA) and 6 times (poly rA) greater than the monomer. Comparison of emission spectra and polarisation spectra demonstrates heterogeneity of emission and, following attempts at spectral synthesis, comparison with experimental reference spectra allows the observed total emission spectra to be resolved into four components assigned as I monomer-like emission, II excimer fluorescence, HI monomer phosphorescence, IV excimer phosphorescence; the polarisation of the excimer fluorescences of ApA and poly rA are deduced. The observations can be understood quantitatively using a simple stacking model with excimer emissions originating in the stacked components. Quantitative differences between ApA and poly rA are due to their different hypochromism and extent of stacking. Differences of polarisation are attributed to different stacking geometries, and the directional properties of the transition moment suggest that the excimer fluorescence is largely charge-resonance in nature.     

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.     

INTERACTION BETWEEN DNA AND 8–METHOXYPSORALEN STUDIED BY LINEAR DICHROISM

Abstract— High sensitivity linear dichroism on DNA oriented by flow is shown to provide conclusive information about the interaction with 8–methoxypsoralen (8–MOP) both in the dark and by irradiation. 8–MOP forms directly in the dark a weak complex with DNA with a structure consistent with the intercalation model of Lerman. When aqueous DNA-psoralen solutions are UV irradiated (30800 nm). a photoadduct is formed. By comparison with experiments in ice matrix, it is suggested that it is the primarily bound 8–MOP that is responsible for the final product. It is inferred that this product is the 4 ¹ ,5 ^I -photoadduct of 8–MOP. The orientation of the photo adduct on DNA is not consistent with intercalation.     

ON THE PHOTOIONIZATION ENERGY THRESHOLD OF TRYPTOPHAN IN AQUEOUS SOLUTIONS

Abstract— To investigate the existence and energy position of a photoionization threshold. tryptophan (Trp) has been photoionized in desecrated neutral aqueous or alcoholic solution under monochromatic light of variable frequency, in presence of N₂O to scavenge the photoelectron.
Present findings and some literature data converge to show the existence of a threshold for the one photon ionization process. This threshold is located at 4.5 ± 0.1 eV and 4.85 ± 0.1 cV for Trp in aqueous and ethanol solutions. respectively, which corresponds to a lowering with respect to the gas phase ionization potential of 3.4 and 3.0 eV.
The photoionization quantum yields for Trp is found about 4 times greater at 250 nm than at Λ_cx= 265 nm, where φe^-_4M=0.080±0.025. In such spectral range. at most one photoelectron out of 4–5 escaping geminate recombination would lead to Trp photodegradation in acrated solutions.
These results also point out that the neutral radical Trp. which has been previously observed for Λ_cx > 275 nm, i.e. below the ionization threshold energy—would not necessarily derive from Trp ⁺ deprotonation or cation-electron dissociative recombination. Similarly, the opening of the indole ring with formylkynurenine (FK) formation which is observed under aerobic conditions and Λ_cx >, 280 nm would not imply an electron attachment on O₂ but reactions such as Trp +³O₂ or Trp*+³O₂ or else
     

1H-INDENYLFURAN AND THIOPHENE DERIVATIVES– A NEW CLASS OF SINGLET OXYGEN SENSITIZERS

Abstract— Photophysical and photochemical properties of 1H-indenylfuran and thiophene derivatives were studied. UV spectra showed that all the compounds tested absorbed UV light in the region 350–380 nm. The fluorescence spectra of 4a-e showed bands in the region 410–470 nm and quantum yields (φ_F) in the range 0.25–0.88. Attempts to calculate the triplet energy of 4a-e failed except for 4c and 4d , which showed E_T= 43–44 kcal mol^-1. These compounds are a new class of singlet oxygen sensitizers. The sensitized reaction of 2,5-dimethylfuran with singlet oxygen was followed showing that 4a and 4e are more reactive than α-terthiophene (4.0 and 2.5 times respectively), while the other compounds show the same reactivity as 1 . This behavior can be explained considering the different intersystem crossing quantum yields of compounds 4a-e . Diazabicyclo[2. 2. 2]octane is a quencher of singlet oxygen in this reaction, while we can exclude superoxide ion formation using the photooxidation of α, α-dimethylstilbene.     

PHOTOADDITION OF CHLORPROMAZINE TO GUANOSINE-5'-MONOPHOSPHATE

Abstrart—The photochemistry of chlorpromazine (CPZ) with guanosine-5'-monophosphate (GMP) was studied as a model for the photoaddition of CPZ to DNA. Irradiation of CPZ with calf thymus DNA produced a product emitting at 520 nm, whereas with GMP emission was at 495 nm. HPLC separation of photolysis mixtures of [³H]CPZ with GMP and [¹⁴C]GMP with CPZ indicated that three photoadducts were formed. One of the adducts fluoresced at 500 nm and appeared to be the product detected but not separated by Fujita et al. (Photochem. Photobiol . 1981, 34 , 101–105). A second adduct emitted at 460 nm, and the third was nonfluorescent. The photoadduct emitting at 500 nm was characterized by UV, fluorescence, and NMR to be an adduct from coupling of the C-8 position of guanine to the C-2 position of the phenothiazine ring of CPZ. The cation radical of CPZ (CPZ ⁺) does not appear to be an intermediate since enzymatically generated CPZ ⁺ formed a product that eluted with a retention time close to that of the photoadducts, but did not emit at 520 nm.     

FLUORESCENCE DETECTION OF AN 8-METHOXYPSORALEN METABOLITE IN HUMAN INTERSTITIAL FLUID

Abstract— A fluorescent method has been used to study the suction blister fluid of human volunteers collected after 8-methoxypsoralen (8-MOP) oral intake. A fluorescent chromophore with spectral characteristics (Λ_max= 390 nm, Λ_max=470nm) distinct from 8-MOP has been detected. Our results suggest the existence of a metabolite form of 8-MOP within the patients's skin prior to any UV irradiation. This form might result in the opening of the4–5' double bond of the 8-MOP molecule.     

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.     

SOLVENT EFFECTS ON THE TAUTOMERIZATION OF 5'-DEOXYPYRIDOXAL. A PHOTOPHYSICAL STUDY

Abstract— Absorption and fluorescence spectra of 5'-deoxypyridoxal (DPL) in various pure solvents and mixtures were recorded both at room temperature and over the range10–65°C. The areas under the absorption bands were analyzed to obtain the mole fraction (f_N, f_z) of two tautomers (the zwitterionic, Z, and neutral, N, forms) in the ground state. The following spectral parameters were determined from the fluorescence spectra: Stokes shift (Δ v ), fluorescence quantum yield of the neutral form (Q_N), fluorescence ratio of the neutral to the zwitterionic form (ø_N/ø_Z) and the rate constant of tautomerization ( k ₁) from Z to N in the excited state. Some of these parameters (f_N, Δ v , Q_N, k ₁) were found to depend on the proton donor character of the solvent, whereas others (ø_N/ø_Z) depended on its dipole moment. Thus, the absorption and fluorescence spectra of DPL allow one to obtain information on the polarity and the concentration of –OH groups on its environment.     

LUMINESCENCE AND PHOTOCHEMISTRY OF 4-THIOURIDINE IN AQUEOUS SOLUTION

Abstract— In bidistilled water, 4-thiouridine (4TU) exhibits a weak unusual luminescence, the quantum yield of which is 3 × 10^-4 at 25°C. The excitation spectrum corresponds well to the 4TU absorption spectrum. The emission lies at longer wavelengths (Λ_max 550 nm) than the 4TU phosphorescence observed at 77 K (Λ_max, 470–480 nm). From the emission signal obtained after an excitation flash of 3 ns half-width, an "apparent" rate constant for the radiative deactivation process, shorter than 5 × 10⁶ s, can be inferred. The 300 K emission is efficiently quenched by halides and by oxygen: quenching involves a long-lived intermediate (⋍ 200 ns).
Clearly the emissive state X is populated through the S₀-S₁ electronic transition π→π* of 4TU. The nature of X cannot be unambiguously determined: it cannot be an excimer but can be either the 4TU triplet state or another chemical state distinct from the 4TU excited singlet or triplet states.
An interesting finding is that the 300 K emission and the ability of 4TU to photoreact are related: they are quenched with the same efficiency by halide anions. This indicates that quenching occurs at the same long-lived intermediate species , which is either a precursor of the emitter or the emitter itself.     

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.     

SPECTRAL AND TIME DEPENDENCE STUDIES OF THE ULTRA WEAK BIOLUMINESCENCE EMITTED BY THE BACTERIUM Escherichia coli

Abstract— Weak luminescence was detected using photon counting equipment, from oxygenated, liquid cultures of Escherichia coli during two stages of its growth cycle. The first period of emission occurred during the exponential phase of growth and comprised a UV(210–330 nm) band and a visible region(450–620 nm) band, the total intensity being (1.65 ± 0.12) x 10³ counts s^-1. The second period of emission occurred during the stationary phase of growth and comprised only a visible region(450–620 nm) band of intensity (8.72 ± 0.15) x 10³ counts s^-1. When the growth temperature was raised from 306.15 to 310.15 K, the above emission intensities were approximately halved, but the spectra were not changed significantly. No luminescence was observed at either temperature when the E. coli was grown anaerobically. The visible region luminescence was attributed to excited carbonyl groups and excited singlet O₂ dimers formed during the decomposition of lipid peroxides. The UV component was tentatively assigned to oxidative side reactions accompanying the synthesis of proteins.     

ACID-BASE EQUILIBRIA OF THE EXCITED SINGLET AND TRIPLET STATES AND THE SEMI-REDUCED FORM OF ACRIDINE ORANGE

Abstract— The acid-base equilibria of the excited singlet and triplet states of acridine orange (AO) were studied by flash-photolysis and fluorometric methods. The dye is a stronger base in the first excited singlet state (pK_s= 13.3) than in the triplet and ground states (pK_r= 10.3: pK_c, = 10.2); acridine orange follows the trend observed with some other heterocyclic compounds, viz. pK_s > pK_r= pK,_c. At room temperature, an anomalous fluorescence occurs from the dye in basic media: the assignment of this emission is discussed.
The semi-reduced dye was studied as a function of pH. In a large pH range (3–14), only the protolytic equilibrium between the cationic (AOH₂⁺) and the neutral (AOH) radicals was observed; the pK value corresponding to this equilibrium was found to be in the range of pH 5–6.