Antioxidants as aromatic amino acid oxidation products

The accumulation of UV photolysis products of amino acids tyrosine and tryptophan, which possess an antioxidant activity, has been studied by the method of luminol-activated chemiluminescence. The amount of antioxidant products was judged by the value of the total antioxidant potential of a UV-irradiated solution, the measure of which was the distance between the peaks of the chemiluminescence curve in the system 2,2'-azo-bis(2-amidinopropane)hydrochloride + luminol in a UV-irradiated and an unirradiated samples (induction period, tau(i)). Simultaneously, the absorption and fluorescence spectra of unirradiared and UV-irradiated amino acid solutions were recorded. It was shown that, upon the exposure of a tryptophan solution to radiation, the accumulation of the fluorescent product N-formyl kynurenine (lambda(em) = 325 nm, lambda(max) = 440 nm) occures, and the curve of its accumulation was similar to the curve of growth of tau(i) photoproducts produced during UV-radiation. When a tyrosine solution was irradiated, the main fluorescent product was dityrosine (lambda(em) = 310 nm, lambda(max) = 415 nm). Nevertheless, the dose dependencies of the formation of dityrosine, and the total antioxidant potential (tau(i)) were completely different. It was found that another product of tyrosine UV-photolysis, dioxyphenylalanine, possessed a pronounced antioxidant activity. It was concluded that the main antioxidants produced under UV-irradiation of tryptophan is formyl kynurenine, and under the irradiation of tyrosine, dioxyphenylalanine.     

Time-resolved fluorescence studies of dityrosine in the outer layer of intact yeast ascospores.

The (time-resolved) fluorescence properties of dityrosine in the outermost layer of the spore wall of Saccharomyces cerevisiae were investigated. Steady-state spectra revealed an emission maximum at 404 nm and a corresponding excitation maximum at 326 nm. The relative fluorescence quantum yield decreased with increasing proton concentration. The fluorescence decay of yeast spores was found to be nonexponential and differed pronouncedly from that of unbound dityrosine in water. Analysis of the spore decay recorded at lambda ex = 323 nm and lambda em = 404 nm by an exponential series (ESM) algorithm revealed a bimodal lifetime distribution with maxima centered at tau 1C = 0.5 ns and tau 2C = 2.6 ns. The relative amplitudes of the two distributions are shown to depend on the emission wavelength, indicating contributions from spectrally different dityrosine chromophores. On quenching the spore fluorescence with acrylamide, a downward curvature of the Stern-Volmer plot was obtained. A multitude of chromophores more or less shielded from solvent in the spore wall is proposed to account for the nonlinear quenching of the total spore fluorescence. Analysis of the fluorescence anisotropy decay revealed two rotational correlation times (phi 1 = 0.9 ns and phi 2 = 30.6 ns) or a bimodal distribution of rotational correlation times (centers at 0.7 ns and 40 ns) when the data were analyzed by the maximum entropy method (MEM). We present a model that accounts for the differences between unbound (aqueous) and bound (incorporated in the spore wall) dityrosine fluorescence. The main feature of the photophysical model for yeast spores is the presence of at least two species of dityrosine chromophores differing in their chemical environments. A hypothetical photobiological role of these fluorophores in the spore wall is discussed: the protection of the spore genome from mutagenic UV light.     

Origin and properties of fluorescence emission from the extrinsic 33 kDa manganese stabilizing protein of higher plant water oxidizing complex

The fluorescence properties of the isolated extrinsic 33 kDa subunit acting as 'manganese stabilizing protein' (MSP) of the water oxidizing complex in photosynthesis was analyzed in buffer solution. Measurements of the emission spectra as a function of excitation wavelength, pH and temperature led to the following results: (a) under all experimental conditions the spectra monitored were found to be the composite of two contributions referred to as '306 nm band' and 'long-wavelength band', (b) the excitation spectra of these two bands closely resemble those of tyrosine and tryptophan in solution, respectively, (c) the spectral shape of the '306 nm band' is virtually independent on pH but its amplitude drastically decreases in the alkaline with a pK of 11.7, (d) the amplitude of the 'long-wavelength' emission band at alkaline pH slightly increases when the pH rises from 7.2 to about 11.3 followed by a sharp decline at higher pH, and (e) the shape of the overall spectrum at pH 7.2 is only slightly changed upon heating to 90 degrees C whereas the amplitude significantly declines. Based on these findings the two distinct fluorescence bands are ascribed to tyrosine(s) ('306 nm band') and the only tryptophan residue W241 of MSP from higher plants ('long-wavelength band') as emitters which are both embedded into a rather hydrophobic environment.     

Tryptophan fluorescence in electron-transfer flavoprotein:ubiquinone oxidoreductase: fluorescence quenching by a brominated pseudosubstrate

We have studied the intrinsic fluorescence of the 12 tryptophan residues of electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO). The fluorescence emission spectrum (lambda ex 295 nm) showed that the fluorescence is due to the tryptophan residues and that the contribution of the 22 tyrosine residues is minor. The emission maximum (lambda m 334 nm) and the bandwidth (delta lambda 1/2 56 nm) suggest that the tryptophans lie in hydrophobic environments in the oxidized protein. Further, these tryptophans are inaccessible to a range of ionic and nonionic collisional quenching agents, indicating that they are buried in the protein. Enzymatic or chemical reduction of ETF:QO results in a 5% increase in fluorescence with no change of lambda m or delta lambda 1/2. This change is reversible upon reoxidation and is likely to reflect a conformational change in the protein. The ubiquinone analogue Q0(CH2)10Br, a pseudosubstrate of ETF:QO (Km = 2.6 microM; kcat = 210 s-1), specifically quenches the fluorescence of one tryptophan residue (Kd = 1.6-3.2 microM) in equilibrium fluorescence titrations. The ubiquinone homologue UQ-2 (Km = 2 microM; kcat = 162 s-1) and the analogue Q0(CH2)10OH (Km = 2 microM; kcat = 132 s-1) do not quench tryptophan fluorescence; thus the brominated analogue acts as a static heavy atom quencher. We also describe a rapid purification for ETF:QO based on extraction of liver submitochondrial particles with Triton X-100 and three chromatographic steps, which results in yields 3 times higher than previously published methods.     

Dimethyl-and monomethyloxyluciferins as analogs of the product of the bioluminescence reaction catalyzed by firefly luciferase

The absorption and fluorescence spectra of dimethyloxyluciferin (DMOL) and monomethyloxyluciferin (MMOL) were studied at pH 3.0-12.0. In the range of pH 3.0-8.0, the fluorescence spectrum of DMOL exhibits a maximum at lambda(em) = 639 nm. At higher pH values an additional emission maximum appears at lambda(em) = 500 nm (wavelength of excitation maximum lambda(ex) = 350 nm), which intensity increases with time. It is shown that this peak corresponds to the product of DMOL decomposition at pH > 8.0. The absorption spectra of MMOL were studied in the range of pH 6.0-9.0. At pH 8.0-9.0, the absorption spectrum of MMOL exhibits one peak at lambda(abs) = 440 nm. At pH 7.3-7.7, an additional band appears with maximum at lambda(abs) = 390 nm. At pH 6.0-7.0 two maxima are observed, at lambda(abs) = 375 and 440 nm. The fluorescence spectra of MMOL (pH 6.0-9.7, lambda(ex) = 440 or 375 nm) exhibit one maximum. It is shown that decomposition of DMOL and MMOL in aqueous solutions results in products of similar structure. DMOL and MMOL are rather stable at the pH optimum of luciferase. It is suggested that they can be used as fluorescent markers for investigation of the active site of the enzyme.     

A comparison of the intrinsic fluorescence of red kangaroo, horse and sperm whale metmyoglobins

Several metmyoglobins (red kangaroo, horse and sperm whale), containing different numbers of tyrosines, but with invariant tryptophan residues (Trp-7, Trp-14), exhibit intrinsic fluorescence when studied by steady-state front-face fluorometry. The increasing tyrosine content of these myoglobins correlates with a shift in emission maximum to shorter wavelengths with excitation at 280 nm: red kangaroo (Tyr-146) emission maximum 335 nm; horse (Tyr-103, -146) emission maximum 333 nm; sperm whale (Tyr-103, -146, -151) emission maximum 331 nm. Since 280 nm excites both tyrosine and tryptophan, this strongly suggests that tyrosine emission is not completely quenched but also contributes to this fluorescence emission. Upon titration to pH 12.5, there is a reversible shift of the emission maximum to longer wavelengths with an increase greater than 2-fold in fluorescence intensity. With excitation at 305 nm, a tyrosinate-like emission is detected at a pH greater than 12. These studies show that: (1) metmyoglobins, Class B proteins containing both tyrosine and tryptophan residues, exhibit intrinsic fluorescence; (2) tyrosine residues also contribute to the observed steady-state fluorescence emission when excited by light at 280 nm; (3) the ionization of Tyr-146 is likely coupled to protein unfolding.     

Fluorescence properties of free and protein bound fluorescein dyes. I. Macrospectrofluorometric measurements.

Excitation and emission properties of fluorescein derivatives were studied macrofluorometrically. Measurements were performed with solutions of various concentrations (0.07-100 microgram/ml) of free sodium fluorescein prepared from fluorescein diacetate (FDA), fluorescein isothiocyanate (FITC) and FITC bound to rabbit gamma-globulin. Both excitation and emission spectra as well as fluorescence intensities at constant excitation/emission wavelengths (496/515 nm) were recorded. The findings indicate that (1) FDA gives about twice the fluorescence intensity compared to equal concentrations of FITC. (2) The fluorescence properties of FITC upon excitation with blue light (lambda = 496 nm) are only slightly altered by the conjugation to rabbit gamma-globulin. (3) Considerable quenching due to conjugation could, however, be shown to occur upon UV excitation (lambda = 340 nm). (4) Fluorescence emission excited by visible blue light (496 nm) increases linearly to dye concentration in a range of 0.07-2.5 microgram/ml. Beginning at 5 microgram/ml (10-(5) M/1) all three compounds show a sharp decrease of fluorescence intensity with further increasing concentration. Practical aspects of these data for the immunofluorescence method are discussed.     

Calcium binding to calmodulin mutants monitored by domain-specific intrinsic phenylalanine and tyrosine fluorescence

Cooperative calcium binding to the two homologous domains of calmodulin (CaM) induces conformational changes that regulate its association with and activation of numerous cellular target proteins. Calcium binding to the pair of high-affinity sites (III and IV in the C-domain) can be monitored by observing calcium-dependent changes in intrinsic tyrosine fluorescence intensity (lambda(ex)/lambda(em) of 277/320 nm). However, calcium binding to the low-affinity sites (I and II in the N-domain) is more difficult to measure with optical spectroscopy because that domain of CaM does not contain tryptophan or tyrosine. We recently demonstrated that calcium-dependent changes in intrinsic phenylalanine fluorescence (lambda(ex)/lambda(em) of 250/280 nm) of an N-domain fragment of CaM reflect occupancy of sites I and II (VanScyoc, W. S., and M. A. Shea, 2001, Protein Sci. 10:1758-1768). Using steady-state and time-resolved fluorescence methods, we now show that these excitation and emission wavelength pairs for phenylalanine and tyrosine fluorescence can be used to monitor equilibrium calcium titrations of the individual domains in full-length CaM. Calcium-dependent changes in phenylalanine fluorescence specifically indicate ion occupancy of sites I and II in the N-domain because phenylalanine residues in the C-domain are nonemissive. Tyrosine emission from the C-domain does not interfere with phenylalanine fluorescence signals from the N-domain. This is the first demonstration that intrinsic fluorescence may be used to monitor calcium binding to each domain of CaM. In this way, we also evaluated how mutations of two residues (Arg74 and Arg90) located between sites II and III can alter the calcium-binding properties of each of the domains. The mutation R74A caused an increase in the calcium affinity of sites I and II in the N-domain. The mutation R90A caused an increase in calcium affinity of sites III and IV in the C-domain whereas R90G caused an increase in calcium affinity of sites in both domains. This approach holds promise for exploring the linked energetics of calcium binding and target recognition.     

Venom exonuclease. IV. Intrinsic fluorescence of the exonuclease from Crotalus adamanteus venom

The intrinsic fluorescence of the exonuclease isolated from Crotalus adamanteus venom, was studied. The position of its maximum at 335 nm and half-width of the emission band 55 nm (lambda exc. 295 nm) suggested the existence of at least two types of tryptophan residues in the enzyme molecule. Differential analysis of the fluorescence spectra obtained by excitation at 280 and 295 nm revealed about 12.5% contribution of the tyrosine fluorescence in the overall emission excited at 280 nm. The environment of the tryptophan residues in the exonuclease was studied by quenching of their fluorescence with various ionic (NO3-, NO2-, I-, Br- and Cs+) and non-ionic agents (acrylamide, chloroform-methanol). On this basis, fractions of inner (non-polar) and surface tryptophan residues located in charged and neutral regions of the enzyme molecule were evaluated. More than half of the residues (60%) was found in the inner part of the exonuclease while most of its surface tryptophans--in a neutral region(s).     

Cofactor and DNA interactions in EcoRI DNA methyltransferase. Fluorescence spectroscopy and phenylalanine replacement for tryptophan 183.

EcoRI DNA methyltransferase contains tryptophans at positions 183 and 225. Tryptophan 225 is adjacent to residues previously implicated in S-adenosylmethionine (AdoMet) binding and to cysteine 223, previously shown to be the site of N-ethyl maleimide-mediated inactivation of the enzyme (Reich, N. O., and Everett, E. (1990) J. Biol. Chem. 265, 8929-8934; Everett, E. A., Falick, A. M., and Reich, N. O. (1990) J. Biol. Chem. 265, 17713-17719). The fluorescence spectra of the wild-type enzyme is centered at 338 nm indicating partial tryptophan solvent accessibility. Substitution of tryptophan 183 with phenylalanine results in a 45% drop in fluorescence intensity, but no shift in lambda max. DNA binding to the wild-type methyltransferase caused an increase in the fluorescence intensity, while binding to the tryptophan 183 mutant had a quenching effect, suggesting that DNA binding induces a conformational change near both tryptophans. Binding of AdoMet and various AdoMet analogs to the wild-type methyltransferase results in no change in the fluorescence spectrum when excitation occurs at 295 nm, suggesting that no conformational change occurs, and AdoMet does not interact with either tryptophan. In contrast, quenching was observed when excitation occurred at 280 nm, suggesting that AdoMet and its analogs may be quenching tyrosine to tryptophan energy transfer. Protein-ligand complexes were titrated with acrylamide, and the data also implicate conformational changes upon DNA binding but not upon AdoMet binding, consistent with previous limited proteolysis results (Reich, N. O., Maegley, K. A., Shoemaker, D.D., and Everett, E. (1991) Biochemistry 30, 2940-2946).     

Isolation and fluorescence studies on a lipophorin from the weevil diaprepes abbreviatus

Lipophorin was isolated from larvae of a root weevil, Diaprepes abbreviatus (Coleoptera: Curculionidae), using density gradient ultracentrifugation. D. abbreviatus lipophorin contained two apoproteins, apolipophorin-I (M_r = 226,000) and apolipophorin-II (M_r = 72,100) and had a density of 1.08. Relative to other larval lipophorins, D. abbreviatus lipophorin contained little cysteine (determined as cysteic acid) and methionine. Fluorescence spectroscopy of intrinsic tyrosine and tryptophan residues excited at 290 nm revealed a single broad emission peak at 330 nm. Upon denaturing and delipidating lipophorin in guanidine HCl, this peak resolved into two peaks with maxima at 305 and 350 nm. Excitation spectra suggested that the two peaks were due to tyrosine and tryptophan, respectively. Fluorescence quenching agents, iodide and acrylamide, were used to determine accessibility of tyrosine and tryptophan residues to the aqueous environment. Iodide, a polar quenching agent, did not quench fluorescent emission from native lipophorin; quenching by iodide increased to moderate levels when lipophorin was denatured in guanidine HCl. Acrylamide quenched the fluorescence of native lipophorin moderately and very efficiently quenched fluorescence of denatured lipophorin. No difference was observed between fluorescence quenching of denatured vs. denatured and delipidated lipophorin by either iodide or acrylamide.     

The fine structure of luminescence spectra of azurin.

The spectra of azurin absorption, fluorescence, phosphorescence and fluorescence excitation have been measured in aqueous solutions at ordinary and liquid nitrogen temperatures. The fluorescence spectra of azurin even at ordinary temperatures have a well resolved fine vibrational structure. The frequency analysis reveals practically the same wave number distances between the main structure peaks in fluorescence spectra at room and low temperatures and in phosphorescence spectra. The comparison of the protein absorption and excitation spectra shows that all the energy absorbed by tyrosine residues is transferred onto indole chromophore. These data suggest an unusual tryptophan environment in this protein, which is characterized by the absence of any hydrogen bonding or other polar interaction of tryptophan with its environment. The problem of the possibility of contributions of two electronic transitions (1La in equilibrium A and 1Lb in equilibrium A) in absorption and emission spectra of azurin tryptophan arising from their mirror symmetry is discussed.     

Aromatic amino acid oxidation products as antioxidants

The accumulation of UV photolysis products of amino acids tyrosine and tryptophan, which possess antioxidant activity, has been studied by the method of luminol-dependent chemiluminescence. The amount of antioxidant products was judged by the value of the total antioxidant potential of a UV-irradiated solution, the measure of which was the distance between the peaks of the chemiluminescence curve in the system 2,2′-azo-bis(2-amidinopropane) hydrochloride + luminol with a UV-irradiated and an unirradiated sample (induction period, τ _i ). Simultaneously, the absorption and fluorescence spectra of unirradiared and UV-irradiated amino acid solutions were recorded. It was shown that exposure of a tryptophan solution to radiation led to accumulation of a fluorescent product N-formyl kynurenine (λ_em = 325 nm, λ_max = 440 nm), and the curve of its accumulation was similar to the growth of antioxidant potential. When a tyrosine solution was irradiated, the main fluorescent product was dityrosine (λ_em = 310 nm, λ_max = 415 nm). Nevertheless, the dose dependences of the formation of dityrosine and the total antioxidant potential were completely different. It was found that another product of tyrosine UV photolysis, dihydroxyphenylalanine, possessed pronounced antioxidant activity. It was concluded that the main antioxidant produced under UV irradiation of tryptophan is formyl kynurenine, and under irradiation of tyrosine it is dihydroxyphenylalanine.     

Luminescence studies of saccharide binding to wheat germ agglutinin (lectin).

The fluorescence and phosphorescence emission of wheat germ agglutinin are reported. Fluorescent tryptophan residues of wheat germ agglutinin are found highly exposed to solvent: fluorescence quenching induced by temperature fits with a single Arrhenius critical energy close to that of tryptophan in solution; the whole fluorescence emission is susceptible to iodide ion quenching and data reveal the homogeneity of fluorescence arising from only one type of tryptophan exposition. Energy transfers are analyzed at singlet and triplet state level. Tyrosine fluorescence at 25 degrees C is very weak. Results obtained from the relative excitation fluorescence quantum yield and from intrinsic fluorescence polarization show that a large amount of energy absorbed by tyrosine at 280 nm is transferred to tryptophan residues. However, tyrosine fluorescence is highly increased at 70 degrees C although disulfide bridges are not reduced. The phosphorescence spectrum at 77 K in 50% ethylene glycol is finely structured with several resolved vibrational bands at 405, 432 and 455 nm. Phosphorescence decay can be fitted with a single exponential. Lifetime is independent of excitation wave-length. Its value is very close to that of free tryptophan. Influence of tri-N-acetyl-chitotriose binding on luminescence properties are investigated. Results are analyzed in terms of steric tryptophan-ligand relationships. It is shown that all the fluorescent chromophores are concerned by the ligand binding but all fluorescence emission is still susceptible to iodide ion quenching. There is no change induced in energy transfer at the singlet state level and no modification in triplet state population.     

Fluorescence studies of the interaction of DNA with Hoechst 33258

Absorption and fluorescence measurements of DNA-Hoechst 33258 complexes at high molar ratio of DNA phosphate to dye are consistent with the existence of two types of bound species. One type (Type I) predominates at high ionic strength, whereas the other (Type II) occurs at low ionic strength. The fluorescence peak (lambda fmax) depends on the excitation wavelength (lambda ex); lambda fmax shifts toward longer wavelength with increasing lambda ex. Optical properties obtained are summarized in the following: for Type I, lambda amax (absorption) = 352 nm, lambda fmax at lambda ex of 335 nm = 460 nm, tau (fluorescence lifetime) = 2.0-2.5 ns; for Type II, lambda amax = 360 nm, lambda fmax at lambda ex of 335 nm = 470 nm, tau = 4.0-5.0 ns. This behavior is interpreted in terms of solvent-solute relaxation. Type I corresponds to less hydrated bound species, while Type II to more hydrated bound species.     

Evaluating UV-B effects and EDU protection in cucumber leaves using fluorescence images and fluorescence emission spectra

A newly developed laboratory fluorescence imaging system was used to obtain fluorescence images (FImage) of freshly excised cucumber (Cucumis sativus L.) leaves in spectral bands centered in the blue (F450), green (F550), red (F680), and far-red (F730) spectral regions that resulted from a broad-band (300-400 nm) excitation source centered at 360 nm. Means of relative fluorescence intensities (RFI) from these spectral fluorescence images were compared with spectral fluorescence emission data obtained from excitation wavelengths at 280 nm (280EX, 300-550 nm) and 380 nm (380EX, 400-800 nm) of dimethyl sulfoxide (DMSO) extracts from these leaves. All three fluorescence data types (FImage, 280EX, 380EX) were used to assess ultraviolet-B (UV-B, 280-320 nm) induced physiological changes and the possible use of N-[2-(2-oxo-1-imidazolidinyl) ethyl]-N′-phenylurea (EDU or ethylenediurea) as a chemical protectant against UV-B damage. Plants exhibited well known foliar growth and pigment responses to UV-B exposure (e.g., increased UV-B absorbing compounds and decreased leaf area, chlorophyll a content; and and lower chlorophyll a/b and chlorophyll/carotenoid pigment ratios). Since EDU alone had no effect on foliar variables, there was no evidence that EDU afforded protection against UV-B. Instead, EDU augmented some UV-B effects when provided in conjunction with UV-B irradiation (e.g., reductions in the chlorophyll/carotenoid ratio, total photosynthetic pigments, and chlorophyll b content).Relative fluorescence intensities (RFI) in the longer visible wavelengths (green, red, and far-red) were uncorrelated for comparisons between the FImage and 380EX data sets. However, blue and green RFI were significantly correlated (0.8r0.6; P ≤0.002) for comparisons between FImage and 280EX data sets. UV-B treatment caused an increase in blue RFI (e.g., F450) in both images and 280EX measurements. One explanation is that the UV-B excitation of both 280EX and FImage stimulates processes that produce excess blue fluorescence. The molecules that produce the excess blue fluorescence in both the 280EX and the Fimage data are different electron transfer agents that operate in parallel. For FImage, the UV excitation penetrates leaf surface layers to stimulate fluorescence from compounds in mesophyll and epidermal tissues (as occurs for the extracts of leaf discs), whereas emissions captured at longer, less energetic wavelengths, were primarily from the epidermal layer. UV-B irradiated leaves showed much greater heteorgeneity of RFI in both the green (F550_FImag) and the red (F680_FImag) bands than unirradiated leaves; this was true irrespective of EDU treatment.Although qualitative responses in individual bands differed between FImage and 380EX data, similar results were obtained in the detection of UV-B induced effects when the red/green and blue/far-red fluorescence ratios of these data were compared. The red/green ratio (either F680/F550_FImage or F675/F525_380EX) was lower for UV-B exposed plants in both images and 380EX data. UV-B exposure also significantly enhanced the blue/far-red ratio of images (F450/F740_FImage) and the comparable 380EX ratio (F450/F730_380EX) for the combined UV-B/EDU group. The far-red/red ratios were not useful in separating treatment effects in images or 380EX. Although comparable ratios were not available in 280EX data, the UV/blue ratio (F315/F420_280EX) was substantially reduced by UV-B exposure and was inversely related to total photosynthetic pigment content. These findings suggest that the red/green ratio (FImage, 380EX) and the UV/blue ratio (280EX) may be as useful as the blue/far-red ratio (380EX) reported previously in detection of UV-B stress. Furthermore, the results support the validity of the imaging technique as a non-destructive diagnostic tool for assessing UV-B stress damage in plants.     

Dual-photon excitation of albumin

Fluorescence emission after two-photon excitation at 580 nm is observed in albumin by means of Nd:YAG laser at room temperature. The two-photon excitation spectral range 550-590 nm was obtained. The experimental results show that albumin fluorescence originates from tryptophan residues.     

Chemiluminescence in L-tyrosine-H2O2-horseradish peroxidase system: possible formation of tyrosine cation radical

Tyrosine-H2O2-horseradish peroxidase system at pH 7.4 emitted light in visible region. Phenolic compounds other than tyrosine were also emissive, whereas methoxy phenylalanine and phenyl compounds were not, in H2O2-peroxidase systems. Chemiluminescence spectrum of tyrosine of tyrosine-H2O2-horseradish peroxidase system showed two prominent peaks at 478 nm and 500 nm (Luminescence 1) and additional two or three peaks near 550 and 610 nm (Luminescence 2). Luminescence 1 is quite similar to the phosphorescence originated from an excited tyrosine in triplet state, while Luminescence 2 is quite similar to the phosphorescence originated from an indole in triplet state. Possible formation of tyrosine cation radical (a precursor of the excited tyrosine) and indole cation radical in the enzyme protein (a precursor of the excited tryptophan residue) were discussed.     

Study of spectral and luminescent manifestations of the aggregation of thymine chromophores in aqueous solutions of polythymidylic acid at room temperature in connection with the task of photochemical record of information on thymine

Luminescence and excitation luminescence spectra of water solutions of polythymidylic acid at room temperature were studied. Three luminescence bands at different excitation wavelengths were observed: at 338 nm, which was known earlier, and two new bands, at 320 and 350 nm. The study of excitation luminescence spectra that have not been studied earlier led us to interpret the band at 320 nm as a band of chromophores that do not interact, the band at 338 nm as a band of photochemically most active densely packed stacking dimers (absorption band exciton splitting approximately 4000 cm(-1)), and the band at 350 nm as a band of photochemically inactive big stacking aggregates (n > or = 10, exciton splitting approximately 8000 cm(-1)). Changes in optical density at 270 nm of poly-T water solutions after consecutive irradiations with UV light at 297+302 and 248 nm were studied. The causes of incomplete reversibility are discussed.     

Protein-flavonol interaction: fluorescence spectroscopic study

Recent studies have shown that various synthetic as well as therapeutically active naturally occurring flavonols possess novel luminescence properties that can potentially serve as highly sensitive monitors of their microenvironments in biologically relevant systems. We report a study on the interactions of bovine serum albumin (BSA) with the model flavonol 3-hydroxyflavone (3HF), using the excited-state proton-transfer (ESPT) luminescence of 3HF as a probe. Upon addition of BSA to the flavonoid solutions, we observe remarkable changes in the absorption, ESPT fluorescence emission and excitation profiles as well as anisotropy (r) values. Complexation of 3HF with protein results in a pronounced shift (20 nm) of the ESPT emission maximum of the probe (from lambda(max)(em) = 513 nm to lambda(max)(em) = 533 nm) accompanied by a significant increase in fluorescence intensity. The spectral data also suggest that, in addition to ESPT, the protein environment induces proton abstraction from 3HF leading to formation of anionic species in the ground state. Fairly high values of anisotropy are observed in the presence of BSA for the tautomer (r = 0.25) as well as anion (r = 0.35) species of 3HF, implying that both the species are located in motion-restricted environments of BSA molecules. Analysis of relevant spectroscopic data leads to the conclusions that two binding sites are involved in BSA-3HF interaction, and the interaction is slightly positively cooperative in nature with a similar binding constant of 1.1 - 1.3 x 10(5) M(-1) for both these sites. Proteins 2001;43:75-81.