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.     

Comparative fluorescence properties of lipoxygenases

1. Lipoxygenases purified from tomato, rat liver and soybean show a fluorescence band centered at 648 nm, which is likely to derive from Tyr and Trp. 2. The intensity of this fluorescence range from 0.7 to 1.0% of the intensity of their major intrinsic fluorescence band (lambda max = 343 nm) in all these lipoxygenases. 3. At inhibitory concentrations, ditizone partly quenches the fluorescence of the lipoxygenases above 600 nm. 4. Saturating concentrations of linoleic acid produce 79% quenching of the fluorescence at 648 nm of soybean lipoxygenase inactivated by treatment with 1 mM dithiothreitol. From these data we have obtained an apparent Kd for linoleic acid-lipoxygenase complex dissociation of 34 +/- 3 microM. 5. It is suggested that the fluorescence above 600 nm reveals the presence of aromatic amino acids located near or at the catalytic center.     

表面活性剂增敏——牛血清白蛋白荧光猝灭法测定槐花中芦丁含量

目的：应用牛血清白蛋白荧光猝灭法建立一种测定槐花中芦丁含量的新方法。方法：牛血清白蛋白（BSA）具有很强的内源荧光性,而芦丁溶液本身不产生荧光。当芦丁与BSA结合后,会导致其荧光强度下降,表面活性剂吐温-80（T-80）对体系有促进荧光猝灭作用。BSA在λex=338nm处的荧光猝灭程度与芦丁的量在一定浓度范围内呈良好的线性关系,据此建立测定槐花中芦丁含量的新方法。结果：该结合物的最大发射波长为λmax=338nm,与芦丁摩尔浓度在6×10-7-3.0×10-5mol.L-1范围内线性关系良好。其线性回归方程为ΔF=136.36CRu（×10-5mol.L-1）-0.5454,相关系数r=0.9976,检出限为1.58×10-7mol.L-1,RSD为2.8%-4.3%,加标回收率为97.6%~101.2%。结论：本方法操作简便、快速,用于实际样本的测定,结果满意。     

Long-wavelength fluorescence of tyrosine and tryptophan solutions

We report in this paper the presence of fluorescence bands of tryptophan and tyrosine solutions centered above 550 nm. This long-wavelength fluorescence is of much lower intensity, (0.4-2.7)%, than the UV fluorescence of these aromatic aminoacids. The basic characteristic of these fluorescence bands are: (a) tyrosine: lambda em = 600 nm with two excitation peaks centered at 453 nm and 550 nm (b) tryptophan: lambda em = 675 nm with two excitation peaks centered at 455 and 560 nm. It has been found that irradiation of tyrosine solutions with a potent UV lamp promotes an important increase of absorption at 310 nm and above 400 nm.     

A coupled fluorometric rate assay for pyruvate dehydrogenase in cultured human fibroblasts

A method for measuring the activity of the pyruvate dehydrogenase complex (PDC) by coupling acetyl-CoA production to acetylation of a fluorescent dye is described. Acetylation of cresyl violet acetate by pigeon liver acetyltransferase results in a shift of its fluorescence spectrum from lambda ex max = 575, lambda em max = 620 nm to lambda ex max = 475, lambda em max = 575 nm. The rate of appearance of acetylated dye was followed fluorometrically and was proportional to PDC activity in extracts of cultured human fibroblasts. The assay showed appropriate substrate and cofactor dependence and had a working range between 0.04 and 70 munits. It is 10 times more sensitive than the spectrophotometric assay on which it is based (working range 0.4-31 munits) and is equally convenient. Unactivated PDC activity in fibroblast extracts was 0.75 (0.60-0.92) munits/mg protein (mean and range for six cell lines).     

Fluorescence resonance energy transfer from tryptophan to a chromium(III) complex accompanied by non-specific cleavage of albumin: a step forward towards the development of a novel photoprotease

A chromium(III) complex, transdiaqua [N, N'-propylenebis(salicylideneimino)chromium(III)]perchlorate ([Cr(salprn)(H2O)(2)]ClO(4)) in the presence of sodium azide and upon photoexcitation was found to bring about non-selective cleavage of bovine serum albumin (BSA). Electron paramagnetic resonance (EPR) evidence has been obtained for the formation of a Cr(V) species upon photolysis of a solution containing the chromium(III) complex and sodium azide. This Cr(V) species non-selectively cleaves BSA. The fluorescence excitation spectrum of BSA-[Cr(salprn)(H2O)(2)]+ adduct showed a band at lambda(max)(ex) nm due to charge transfer transition of the chromium(III) complex as well as a prominent band at 290 nm attributable to tryptophan absorption. This indicated an efficient Forster type fluorescence energy transfer (FRET) from the tryptophan residues to the chromium(III) complex indicating that the Cr(III) complex binds in the vicinity of the tryptophan residue.     

Intrinsic fluorescence, difference spectrophotometry and theoretical studies on tertiary structure of calf thymus histone H1

Tyr-72 is included in the hydrophobic cleft which is formed in the histone H1 globular head. Tyr-72 is screened against polar aqueous environment and its intramolecular mobility is sharply retarded. This microenvironment causes a red shift (lambda max = 279 nm) and a sharpening of the longer wavelength shoulder of absorption spectra, a high fluoresence anisotropy value (A = 0,11), high quantum yield of fluoresence (approximately 0.2) and a decrease of the Stern-Volmer Constant during quenching of histone H1 fluorescence by acrylamide. It has been found that the change in the intensity of histone fluorescence at lambda excit = 265 nm, but not at lambda excit = 280 nm, is due to the changes in the quantum yield of fluorescence. The increase of fluorescence intensity at lambda excit = 280 nm depends on the changes in the quantum yield and molar extinction coefficient of histone H1 tyrosyl chromophore. The change in the ratio of fluorescence intensity exited at 280 nm (F280) to the fluorescence intensity excited at 265 nm (F265) corresponds to the change of delta epsilon 286 in difference absorption spectra. The introduction of the parameter Cf = F280/F265 allows one to go over to studying excitation spectrum shifts instead of histone absorption spectrum shifts, which is much more convenient methodologically since in this case it is possible to carry out research using lower protein concentrations and turbid solutions. The results make it possible to designate Tyr-72 of histone H1 as a special class of fluorescent tyrosyls whose properties differ from those of tyrosyls of other tryptophane-free proteins: RNAase, insulin, core histones--H2A, H2B, H3, H4 and some others.     

Comparative study of the structural characteristics of aldolase in rabbit muscles in normal states and in alloxan diabetes

It is shown that the activity of aldolase synthesized in rabbit muscles under diabetes is higher than that at normal state. This fact is probably a result of some structural alterations in NAD-binding site with Trp-291 and -311 in it which overlaps a considerable part of C-terminal region of the protein. The hydrophobic part of the enzyme containing Trp-147 under diabetes seems to remain unaltered. This consideration is based on the longwave shift in aldolase fluorescence lambda max (from 320 to 324 nm) under this pathology, suggesting a transition of Trp-291 and -311 into more polar environment and is confirmed by the disappearance of the difference in lambda max in the NADH presence. The NADH-originated shift in lambda max position for the both proteins ended at the same wave-length at 314 nm. The position of lambda max at 324 nm resulting from possible structural modification of NAD-binding site under diabetes correlates with an increase in the Stern-Volmer quenching constant value (from 4359 to 7500 M-1 for aldolase under normal and diabetic states, respectively). These quenching data evidence in favour of the suggestion on the existence of two classes of tryptophanyls in the aldolase molecule.     

Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications.

The synthesis, proof of structure, and the absorption and fluorescence properties of two new unsymmetrical cyanine dyes, thiazole orange dimer (TOTO; 1,1'-(4,4,7,7-tetramethyl-4,7- diazaundecamethylene)-bis-4-[3-methyl-2,3-dihydro-(benzo-1,3-thiaz ole)-2- methylidene]-quinolinium tetraiodide) and oxazole yellow dimer (YOYO; an analogue of TOTO with a benzo-1,3-oxazole in place of the benzo-1,3-thiazole) are reported. TOTO and YOYO are virtually non-fluorescent in solution, but form highly fluorescent complexes with double-stranded DNA (dsDNA), up to a maximum dye to DNA bp ratio of 1:4, with greater than 1000-fold fluorescence enhancement. The dsDNA-TOTO (lambda max 513 nm; lambda maxF 532 nm) and dsDNA-YOYO (lambda max 489 nm; lambda maxF 509 nm) complexes are completely stable to electrophoresis on agarose and acrylamide gels. Mixtures of restriction fragments pre-labeled with ethidium dimer (EthD; lambda maxF 616 nm) and those pre-labeled with either TOTO or YOYO were separated by electrophoresis. Laser excitation at 488 nm and simultaneous confocal fluorescence detection at 620-750 nm (dsDNA-EthD emission) and 500-565 nm (dsDNA-TOTO or dsDNA-YOYO emission) allowed sensitive detection, quantitation, and accurate sizing of restriction fragments ranging from 600 to 24,000 bp. The limit of detection of dsDNA-TOTO and YOYO complexes with a laser-excited confocal fluorescence gel scanner for a band 5-mm wide on a 1-mm thick agarose gel was 4 picograms, about 500-fold lower than attainable by conventional staining with ethidium bromide.     

Detection of neoplastic cells in the bloodstream]

A study was made of the efficacy of trypan blue, acridine orange, tetracycline and oxytetracycline for detection of tumour cells injected into the blood stream of rats. The cells were identified in the mesenteric microvessels by intravital microscopy. Fluorescence of fluorochromized cells was observed in the blue-violet (lambda max = 400 nm) and ultra-violet (lambda max = 365 nm) irradiation of the fluorescent lamp and in the laser irradiation (lambda = 337 nm). The cells stained with acridine orange had a higher fluorescence intensity and a more distinct structure than those labelled with tetracyclines. Identification of cells with trypan blue was more difficult. The fluorescent method of determination is rather simple and permits to indentify tumour cells directly in the blood stream.     

Unfolding of the regulatory subunit of cAMP-dependent protein kinase I.

The unfolding of the recombinant regulatory subunit of cAMP-dependent protein kinase I was followed by monitoring the intrinsic protein fluorescence. Unfolding proceeds in at least two stages. First, the quenching of fluorescence due to cAMP binding is abolished at relatively low levels of urea (less than 2 M) and is observed as an increase in intensity at 340 nm. The high-affinity binding of cAMP is retained in 3 M urea even though the quenching is lost. The second stage of unfolding, presumably representing unfolding of the polypeptide chain, is seen as a shift in lambda max from 340 to 353 nm. The midpoint concentration, Cm, for this process is 5.0 M. Cyclic AMP binding activity is lost at a half-maximal urea concentration of 3.5 M and precedes the shift in lambda max. Unfolding of the protein in the presence of urea was fully reversible; furthermore, the presence of excess levels of cAMP stabilized the regulatory subunit. A free energy value (delta GDH2O) of 7.1 +/- 0.2 kcal/mol was calculated for the native form of the protein when denaturation was induced with either urea or guanidine hydrochloride. Iodide quenching of tryptophan fluorescence was used to elucidate the number of tryptophan residues accessible during various stages of the unfolding process. In the native cAMP-bound form of the regulatory subunit, only one of the three tryptophans in the regulatory subunit is quenched by iodide while more than two tryptophans can be quenched with iodide in the presence of 3 M urea.     

Assembly of Pigment–Protein Complexes in Carotenoid-Deficient Membranes of Plastids from Wheat Seedlings Treated with Norflurazon

The pyridazinone-type herbicide norflurazon SAN 9789 inhibiting the biosynthesis of long-chain carotenoids results in significant decrease in PS II core complexes and content of light-harvesting complex (LHC) polypeptides. At the same time, early light-induced proteins (ELIP) with molecular masses of 20.5-16.5 and 13.5 kD disappear in norflurazon-treated seedlings grown under intermittent (pulsed) light, confirming the hypothesis that they are carotenoid-binding proteins. Full disappearance of Chl a forms at 668, 676, and 690 nm and a sharp decrease in Chl b form at 648 nm in treated seedlings grown under 30 or 100 lx light intensity shows close contact of these forms with carotenoids in the thylakoid membrane. The band shift from 740 to 720 nm in the low-temperature fluorescence spectrum (77 K) suggests a disturbance of energy transfer from LHC to the Chl a form at 710-712 nm.     

Reversible denaturation of Aequorea green-fluorescent protein: physical separation and characterization of the renatured protein

The green-fluorescent protein (GFP) that functions as a bioluminescence energy transfer acceptor in the jellyfish Aequorea has been renatured with up to 90% yield following acid, base, or guanidine denaturation. Renaturation, following pH neutralization or simple dilution of guanidine, proceeds with a half-recovery time of less than 5 min as measured by the return of visible fluorescence. Residual unrenatured protein has been quantitatively removed by chromatography on Sephadex G-75. The chromatographed, renatured GFP has corrected fluorescence excitation and emission spectra identical with those of the native protein at pH 7.0 (excitation lambda max = 398 nm; emission lambda max = 508 nm) and also at pH 12.2 (excitation lambda max = 476 nm; emission lambda max = 505 nm). With its peak position red-shifted 78 nm at pH 12.2, the Aequorea GFP excitation spectrum more closely resembles the excitation spectra of Renilla (sea pansy) and Phialidium (hydromedusan) GFPs at neutral pH. Visible absorption spectra of the native and renatured Aequorea green-fluorescent proteins at pH 7.0 are also identical, suggesting that the chromophore binding site has returned to its native state. Small differences in far-UV absorption and circular dichroism spectra, however, indicate that the renatured protein has not fully regained its native secondary structure.     

Excitation energy transfer from phycobiliprotein to chlorophyll d in intact cells of Acaryochloris marina studied by time- and wavelength-resolved fluorescence spectroscopy.

The fluorescence decay spectra and the excitation energy transfer from the phycobiliproteins (PBP) to the chlorophyll-antennae of intact cells of the chlorophyll (Chl) d-dominated cyanobacterium Acaryochloris marina were investigated at 298 and 77 K by time- and wavelength-correlated single photon counting fluorescence spectroscopy. At 298 K it was found that (i) the fluorescence dynamics in A. marina is characterized by two emission peaks located at about 650 and 725 nm, (ii) the intensity of the 650 nm fluorescence depends strongly on the excitation wavelength, being high upon excitation of phycobiliprotein (PBP) at 632 nm but virtually absent upon excitation of chlorophyll at 430 nm, (iii) the 650 nm fluorescence band decayed predominantly with a lifetime of 70 +/- 20 ps, (iv) the 725 nm fluorescence, which was observed independent of the excitation wavelength, can be described by a three-exponential decay kinetics with lifetimes depending on the open or the closed state (F(0) or F(m)) of the reaction centre of Photosystem II (PS II). Based on the results of this study, it is inferred that the excitation energy transfer from phycobiliproteins to Chl d of PS II in A. marina occurs with a time constant of about 70 ps, which is about three times faster than the energy transfer from the phycobilisomes to PS II in the Chl a-containing cyanobacterium Synechococcus 6301. A similar fast PBP to Chl d excitation energy transfer was also observed at 77 K. At 77 K a small long-lived fluorescence decay component with a lifetime of 14 ns was observed in the 640-700 nm spectral range. However, it has a rather featureless spectrum, not typical for Chl a, and was only observed upon excitation at 400 nm but not upon excitation at 632 and 654 nm. Thus, this long-lived fluorescence component cannot be used as an indicator that the primary PS II donor of Acaryochloris marina contains Chl a.     

Characteristics of the tertiary structure of histone H1 from the calf thymus

By optical methods it has been previously shown that the globular "head" of histone H1 forms a hydrophobic cavity containing Tyr72. The latter is screened from the polar water surrounding and its intramolecular mobility is drastically hindered. As a consequence of the alteration in the micromilieu are a long wave shift (lambda max = 279,5 nm) and a more pronounced longwave absorption spectra, higher anisotropy (A = 0,11), augmented quantum yield of fluorescence (approximately 0,2) and a decrease of the Stern-Volmer constant for Hl at fluorescence quenching by acrylamide. It was found that changes in fluorescence intensity of histones are connected with alterations in the quantum yield of fluorescence at lambda exc = = 265 nm, but not at lambda exc = 280 nm. The changes in fluorescence intensity at light excitation 280 nm (F280) and 265 nm (F265) are in good accordance with shift delta E286 in differential absorption spectra. Introduction of parameter Cf = F280/F265 allows to study shifts of excitation spectra instead of shifts in absorption spectra of histones. This method has certain advantages, since it permits investigations with lower protein concentrations and in turbid solutions. The data obtained allow to draw out Tyr72 of histone Hl into a special class of fluorescent-tyrosyls, that differ in properties from those of other tryptophandevoided proteins: RNAse, insulin and core-histones H2A, H2B, H3 and H4.     

Intrinsic tryptophan fluorescence identifies specific conformational changes at the actomyosin interface upon actin binding and ADP release.

The helix-loop-helix (A-site) and myopathy loop (R-site) are located on opposite sides of the cleft that separates the proposed actin-binding interface of myosin. To investigate the structural features of the A- and R-sites, we engineered two mutants of the smooth muscle myosin motor domain with the essential light chain (MDE), containing a single tryptophan located either in the A-site (W546-MDE) or in the R-site (V413W MDE). W546- and V413W-MDE display actin-activated ATPase and actin-binding properties similar to those of wild-type MDE. The steady-state fluorescence properties of W546-MDE [emission peak (lambda(max)) = 344, quantum yield = 0.20, and acrylamide bimolecular quenching constant (k(q)) = 6.4 M(-)(1). ns(-)(1)] and V413W-MDE [lambda(max) = 338, quantum yield = 0.27, and k(q) = 3.6 M(-)(1).ns(-)(1)] demonstrate that Trp-546 and Trp-413 are nearly fully exposed to solvent, in agreement with the crystallographic data on these residues. In the presence of actin, Trp-546 shifts to a more buried environment in both the ADP-bound and nucleotide-free (rigor) actomyosin complexes, as indicated by an average lambda(max) of 337 or 336 nm, respectively, and protection from dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide (DHNBS) oxidation. In contrast, Trp-413 has a single conformation with an average lambda(max) of 338 nm in the ADP-bound complex, but in the rigor complex it is 50% more accessible to DHNBS oxidation and can adopt a range of possible conformations (lambda(max) = 341-347 nm). Our results suggest a structural model in which the A-site remains tightly bound to actin and the R-site adopts a more flexible and solvent-exposed conformation upon ADP release.     

Effect of gamma-irradiation on H3 histone and DNA in solution

Three methods, namely, absorbance of colour by reaction with Folin-Ciocalteau reagent, UV absorbance and fluorescence intensity measurements for detection of H3 histone in 0.15 M standard saline citrate (SSC) solution were compared. Maximum sensitivity was found with the Folin-Ciocalteau method. Effect of varying pH and of gamma- radiation on H3 histone and on interaction of H3 histone with DNA were studied. For this, solutions of H3 histone in SSC, in 0.9% NaCl, H3 histone + DNA in 0.9% NaCl were subjected to varying pH (1-10) and gamma- radiation (dose 10-50 Gy) and lambda(max) and Alambda(max) were monitored. From the molar ratios of histone and DNA in the complex, it was observed that at gamma -radiation dose of 50 Gy and pH 8.54, there was a depletion of 6-8 microg/ml of histone from the histone-DNA complex.     

Ferriprotoporphyrin IX mediated oxygen activation/insertion reactions: the anerobic reduction of the aniline-Fe3+-microperoxidase-8 complex by NADH

A spectrophotometric study of the reduction of the Fe3+ microperoxidase-8-aniline (Fe3+-MP-8-An) complex has been carried out. Addition of NADH to a solution of Fe3+-MP-8-An under strictly anerobic conditions results in the formation of a species with lambda max = 414 nm (Fe3+-MP-8-An lambda max 407 nm). The kinetics of formation of this species show an induction period (tau) which follows saturation kinetics with respect to [aniline] with Km(app) = 2.2 x 10(-3) mol dm-3, i.e., close to that obtained in the preceding paper from O2 consumption kinetics mediated by MP-8. Addition of an anerobic solution of the NADH reduced MP-8-An complex, to a saturated O2 solution at pH 12 in the presence of 0.5 mM NADH and aniline 10 mM results in the virtual elimination of the induction phase, which has previously characterized O2 consumption kinetics in ferriprotoporphyrin IX oxygen activation systems. The Arrhenius activation energy for the reduction of the Fe3+-MP-8-An complex is close to that observed for the first reductive step in the cyt P-450 O2 activation cycle. Anerobic reduction of Fe3+-MP-8 by sodium dithionite in 20% MeOH/Aq at pH 8 followed by anerobic titration of the Fe2+-MP-8 (lambda max 420.5 nm) with aniline at pH 12 gives rise to a species lambda max 415 with KD for the process = 4.4 x 10(-3) mol dm-3 (+/- 1.2 x 10(-3) mol dm-3).     

Optical properties of chlorophyll <Emphasis Type="Italic">a</Emphasis> and β-Carotene mixtures adsorbed on bovine serum albumin

The fluorescence of β-carotene/chlorophyll a mixtures in complex with bovine serum albumin in water solution was found to exceed that of one-pigment complexes, being maximal at initial Car/Chl molar ratios of 8–4, whereas the amount of pigment adsorbed on BSA was maximal for an equimolar mixture. The fluorescence spectra of Car-BSA and Chl-BSA complexes largely overlapped (maxima at 684 and 690 nm, respectively).     

Purification of the yellow fluorescent protein from Vibrio fischeri and identity of the flavin chromophore

A low molecular weight protein (approximately 25,000 D) exhibiting a yellow fluorescence emission peaking at approximately 540 nm was isolated from Vibrio fischeri (strain Y-1) and purified to apparent homogeneity. FMN is the chromophore, but it exhibits marked red shifts in both the absorption (lambda max = 380, 460 nm) and the fluorescence emission. When added to purified luciferase from the same strain, which itself catalyzes an emission of blue-green light (lambda max approximately 495 nm), this protein induces a bright yellow luminescence (lambda max approximately 540 nm); this corresponds to the emission of the Y-1 strain in vivo. This yellow bioluminescence emission is thus ascribed to the interaction of these two proteins, and to the excitation of the singlet FMN bound to this fluorescent protein.