Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Fluorometry”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,387 records · Page 77Linked to original sources
PubMed1981

Copper(II).bleomycin, iron(III).bleomycin, and copper(II).phleomycin: comparative study of deoxyribonucleic acid binding.

The kinetics and mechanism of binding of Cu-(II).bleomycin, Fe(III).bleomycin, and Cu(II).phleomycin to DNA were studied by using fluorometry, equilibrium dialysis, electric dichroism, and temperature-jump and stopped-flow spectrophotometry. The affinity of Cu(II).bleomycin for DNA was greater than that of metal-free bleomycin but less than that of Fe(III).bleomycin. Cu(II).bleomycin exhibited a two-step binding process, with the slow step indicating a lifetime of 0.1 s for the Cu(II).bleomycin.DNA complex. Fe(III).bleomycin binding kinetics indicated the presence of complexes having lifetimes of up to 22 s. DNA was lengthened by 4.6 A/molecule of bound Cu(II).bleomycin and by 3.2 A/bound Fe(III).bleomycin but not at all by Cu(II).phleomycin, suggesting that both bleomycin complexes intercalate while the phleomycin complex does not. However, phleomycin exhibited nearly the same specificity of DNA base release as bleomycin. These results suggest that the coordinated metal ion plays a major role in the binding of metal-bleomycin complexes to DNA but that intercalation is neither essential for DNA binding and degradation nor primarily responsible for the specificity of DNA base release by these drugs.

Animals↗
PubMed1979

Differential polarized phase fluorometric investigations of diphenylhexatriene in lipid bilayers. Quantitation of hindered depolarizing rotations.

Differential polarized phase fluorometry has been used to investigate the depolarizing rotations of 1,6-diphenyl-1,3,5-hexatriene (DPH) in isotropic solvents and in lipid bilayers. For DPH dissolved in isotropic solvents, there is a precise agreement between the observed and predicted values for maximum differential tangents, indicating that in these media DPH is a free isotropic rotator. In lipid bilayers the tangent defects (i.e., the differences between the calculated and the observed maximum differential tangents) are too large to be explained by anisotropy in the depolarizing rotations but are accounted for by hindered isotropic torsional motions for the fluorophore [Weber, G (1978) Acta Phys. Pol A 54, 173]. This theory describes the depolarizing rotations of the fluorophore by its rotational rate R (in radians/second) and the limiting fluorescence anisotropy (r) at times long compared with the fluorescence lifetime. Through the combined use of both steady-state anisotropy measurements and differential phase measurements, we have demonstrated that one may obtain unique solutions for both R and r. For DPH embedded in vesicles prepared from dimyristoyl-, dipalmitoyl-, and distearoylphosphatidylcholines, the depolarizing motions are highly hindered at temperatures below the transition temperature (Tc) but are unhindered above Tc. The apparent rotational rates of the probe do not change significantly at Tc. These data suggest that the changes observed in the steady-state anisotropy near Tc derive primarily from changes in the degree to which the probe's rotations are hindered, and only to a small extent from changes in rotational rate. For DPH embedded in bilayers that contained 25 mol % cholesterol, no clear transition occurred and the rotations appeared to be hindered at all temperatures. The rotational motions of DPH embedded in dioleolyphosphatidylcholine were found to be far less hindered, but the rotational rates were similar to those obtained in the saturated phosphatidylcholines. Finally, the data show that in an anisotropic environment, such as that of a lipid bilayer, steady-state fluorescence anisotropy measurements alone cannot yield quantitatively meaningful rotational rates. Extrapolation of steady-state aniosotropy data to the quantitation of membrane viscosity is therefore difficult, if not invalid; however, qualitative comparisons can be useful.

Fluorescence Polarization↗
PubMed1979

On the structure and conformational dynamics of yeast phenylalanine-accepting transfer ribonucleic acid in solution.

The solution structure of yeast tRNAPhe was investigated by using ethidium as a fluorescent probe in the D loop and the anticodon loop. For this purpose the dihydrouracils in position 16/17 and wybutine in position 37 were substituted by ethidium. The lifetimes and the time-dependent anisotropy of ethidium fluorescence were measured by pulsed nanosecond fluorometry. The kinetics of the transitions between different states of the tRNAPheEtd derivatives were determined by chemical relaxation measurements. It was found that the ethidium label irrespective of its position exhibits three different states called T1, T2 and T3 characterized by lifetimes tau 1 = 30 ns, tau 2 = 12 ns, and tau 3 = 3 ns. The lifetime differences are due to different accessibilities of ethidium for solvent quenching in the three states. Thus, there are three different defined structural environments of the ethidium in both the anticodon and the D loop. The distribution of the three states was measured as a function of Mg2+ concentration and temperature; it was found that state T3 is favored over states T2 and T1 by both increasing Mg2+ concentration and increasing temperature. The chemical relaxation kinetics exhibit a fast transition between T1 and T2 (10--100 ms) and a slow transition between T2 and T3 (100--1000 ms). The rates of both transitions depend likewise on Mg2+ concentration and temperature. The equilibrium and kinetic data clearly show the presence of strong and weak interactions between Mg2+ and tRNA. A cooperative model accounting for this behavior is developed. The ethidium probe behaves identically when located in different regions of the tRNA regarding both its distribution of states and its transition kinetics. This suggests that the different spectroscopic states report different conformations of the tRNA structure. The dependence of the three states on Mg2+ and spermine indicates that conformation T3 is closely related to or identical with the crystal structure. The rotational diffusion constants indicate that of all three states T3 is most extended while T2 is most compact. The thermodynamic analysis reveals that the strongly bound Mg2+ ions reduce both the activation entropy and enthalpy of all transitions. The weakly bound Mg2+ ions increase both the activation enthalpy and entropy of the slow transition between T2 and T3. It is suggested that the breaking of several intramolecular bonds, e.g., hydrogen bonds, is involved in this transition.

Ethidium↗
PubMed1977

Energetics of the cooperative and noncooperative binding of nicotinamide adenine dinucleotide to yeast glyceraldehyde-3-phosphate dehydrogenase at pH 6.5 and pH 8.5. Equilibrium and calorimetric analysis over a range of temperature.

The binding of nicotinamide adenine dinucleotide (NAD+) to yeast glyceraldehyde-3-phosphate dehydrogenase (GPDH) has been studied at pH 6.5 and 8.5, at 5,25, and 40 degrees C, by calorimetry, fluorometry, spectrophotometry, equilibrium dialysis, and flow dialysis. As reported earlier for pH 7.3 (Velick S.F., Baggott, J.P., and Sturtevant, J.M. (1971), Biochemistry 10, 779), the binding is accompanied by enthalpy changes which become rapidly more negative as the temperature increases, with delta Cp = -500 to -750 cal deg-1 (mole of NAD+ bound)-1, and by entropy changes which also, as required by the large negative delta Cp, become rapidly more negative with increasing temperature. The binding data at pH 6.5 can be fitted on the basis of either four identical noninteracting sites, or of four sites showing a small degree of negative cooperativity. The data at pH 8.5, particularly at 40 degrees C, require the introduction of positive cooperativity, as was previously shown by Kirschner et al. (Kirschner, K., Eigen, M., Bittman, R., and Voigt, B. (1966), Proc. Natl. Acad. Sci. U.S.A. 56, 1661), and can be equally well fitted on the basis of a sequential model (Adair, G.S. (1925), J. Biol. Chem. 63, 529) or a concerted model (Monod, J., Wyman, J., and Changeux, J.P. (1965), J. Mol. Biol. 12, 88). It is proposed that the observed thermodynamic changes are largely the result of a hydrophobic effect due to a decrease in the exposure of nonpolar groups to the solvent, and of a tightening of the protein structure when the coenzyme is bound with concomitant decrease in the number of easily excitable internal degrees of freedom.

Binding Sites↗
PubMed2002

Binding of discoidin domain receptor 2 to collagen I: an atomic force microscopy investigation.

Collagens have recently been identified as ligands for discoidin domain receptors (DDR1 and DDR2), generating an interest in studying the properties of binding of DDR to its ligand. We are interested in the interaction of DDR2 with collagen I because of its potential role in liver fibrosis. Our in vitro binding assay utilizes DDR2-Fc fusion proteins, which can be clustered (multimerized) by use of antibodies to form DDR2 complexes. Binding of DDR2 complexes to collagen I coated on plastic plates was established by a microplate-based assay using Eu(3+)-labeled proteins and time-resolved fluorometry. Clustering of the DDR2-Fc with antibody was found to be requisite for binding to collagen in vitro. Using atomic force microscopy (AFM) in an aqueous environment, we characterized the surface topographies of DDR2 complexes and collagen I, and investigated binding of this receptor-ligand pair. We were able to image and identify binding of DDR2 complexes onto individual molecules of triple-helical collagen and provide insight into the number and locations of binding sites on collagen I. In most cases, a single receptor complex bound to a single collagen molecule and there were preferred DDR2 binding sites on the collagen I triple helix. These data were validated by rotary-replication transmission electron microscopy (TEM) of glycerol-sprayed samples.

Animals↗
PubMed2003

Interaction of the mitotic inhibitor monastrol with human kinesin Eg5.

The microtubule-dependent kinesin-like protein Eg5 from Homo sapiens is involved in the assembly of the mitotic spindle. It shows a three-domain structure with an N-terminal motor domain, a central coiled coil, and a C-terminal tail domain. In vivo HsEg5 is reversibly inhibited by monastrol, a small cell-permeable molecule that causes cells to be arrested in mitosis. Both monomeric and dimeric Eg5 constructs have been examined in order to define the minimal monastrol binding domain on HsEg5. NMR relaxation experiments show that monastrol interacts with all of the Eg5 constructs used in this study. Enzymatic techniques indicate that monastrol partially inhibits Eg5 ATPase activity by binding directly to the motor domain. The binding is noncompetitive with respect to microtubules, indicating that monastrol does not interfere with the formation of the motor-MT complex. The binding is not competitive with respect to ATP. Both enzymology and in vivo assays show that the S enantiomer of monastrol is more active than the R enantiomer and racemic monastrol. Stopped-flow fluorometry indicates that monastrol inhibits ADP release by forming an Eg5-ADP-monastrol ternary complex. Monastrol reversibly inhibits the motility of human Eg5. Monastrol has no inhibitory effect on the following members of the kinesin superfamily: MC5 (Drosophila melanogaster Ncd), HK379 (H. sapiens conventional kinesin), DKH392 (D. melanogaster conventional kinesin), BimC1-428 (Aspergillus nidulans BimC), Klp15 (Caenorhabditis elegans C-terminal motor), or Nkin460GST (Neurospora crassa conventional kinesin).

Adenosine Diphosphate↗
PubMed2004

Estimation of helix-helix association free energy from partial unfolding of bacterioopsin.

To obtain thermodynamic information about interactions between transmembrane helices in integral membrane proteins, partial unfolding of bacterioopsin in ethanol/water mixtures was studied by Förster-type resonance energy transfer (FRET) from tryptophan to a dansyl group on Lys 41. Tryptophan to dansyl FRET was detected by measuring sensitized emission at 490-500 nm from 285 nm excitation. FRET was observed in dansylbacterioopsin in apomembranes and in detergent micelles but not in 90% ethanol/water or in the chymotrypsin fragment C2 (residues 1-71). The main fluorescence donors are Trp 86 and Trp 182. Increase of FRET from C2 with added chymotrypsin fragment C1 (residues 72-248) provides an estimate of the C1-C2 association constant as 7.7 x 10(6) M(-1). With increasing ethanol concentration, the FRET signal from dansylbacterioopsin in detergent micelles disappeared with a sharp transition above 60% ethanol. No transition occurred in Trp fluorescence from bacterioopsin lacking the dansyl acceptor, nor did dansyl model compounds undergo a similar transition. Light scattering measurements show that the detergent micelles dissipate below 50% ethanol. Thus the observed transition is likely to be a partial unfolding of bacterioopsin. Assuming a two-state unfolding model, the free energy of unfolding was obtained by extrapolation as 9.0 kcal/mol. The slope of the transition (m-value) was -0.8 kcal mol(-1) M(-1). The unfolding process probably involves dissociation of several helices. The rate of association was measured by stopped-flow fluorometry. Two first-order kinetic processes were observed, having approximately equal weights, with rate constants of 2.32 s (-1) and 0.185 s(-1).

Bacteriorhodopsins↗
PubMed2005

Kinetic and thermodynamic studies of tet repressor-tetracycline interaction.

Stopped-flow measurements have been employed to study the kinetics of the conformational changes in TetR (B) induced by tetracycline binding with and without Mg(2+) ions. Result of stopped-flow fluorometry measurements at pH 8.0 indicate conformational changes in the helix-turn-helix motif in the N-terminal domain and in the C-terminal inducer binding domain. Binding of tetracycline (Tc) to TetR in the absence of Mg(2+) can be described by a simple kinetics process, which is limited to the first step association without any unimolecular conformational change step upon Tc binding. The rate constants for this process are equal to 2.0 x 10(5) M(-)(1) s(-)(1) and 2.1 s(-)(1) for the forward and backward reaction, respectively, and gave the binding constant K(a) = 0.96 x 10(5) M(-)(1). The kinetics of [Tc-Mg](+) binding to TetR can be described by reactions in which the first step describes the association characterized by the rate constants k(a) = 1.4 x 10(5) M(-)(1) s(-)(1) and k(d) = 2.2 x 10(-)(2) s(-)(1) and binding constant K(a) = 6.3 x 10(6) M(-)(1). The first step of [Tc-Mg](+) association is followed by at least three conformational change steps, which occur in the inducer binding site and then propagate to the surroundings of Trp75 and Trp43 residues. The rate constants for the forward, k(c), and backward, k(-)(c), reaction for each of these conformational steps have been determined. The thermodynamics of the binding of tetracycline with and without Mg(2+) to TetR was investigated by isothermal titration calorimetry (ITC) at pH 8.0 and 25 degrees C. The measurement shows that TetR dimer possesses two equivalent binding sites for tetracycline, characterized by binding constant K(a) = 9.0 x 10(6) M(-)(1) and K(a) = 7.0 x 10(4) M(-)(1) for Tc with and without Mg(2+), respectively. The binding of the inducer to TetR, in the presence and absence of Mg(2+) ion, is an enthalpy-driven reaction characterized by DeltaH = -51 kJ mol(-)(1) and DeltaH = -33 kJ mol(-)(1), respectively. The entropy change, DeltaS, for the interaction in the presence of Mg(2+) is equal to -38.9 J K(-)(1) mol(-)(1), and for the tetracycline alone, it was estimated at -17.6 J K(-)(1) mol(-)(1).

Kinetics↗
PubMed2006

Deletion of all cysteines in tachyplesin I abolishes hemolytic activity and retains antimicrobial activity and lipopolysaccharide selective binding.

Tachyplesin I is a cyclic beta-sheet antimicrobial peptide isolated from the hemocytes of Tachypleus tridentatus. The four cysteine residues in tachyplesin I play a structural role in imparting amphipathicity to the peptide which has been shown to be essential for its activity. We investigated the role of amphipathicity using an analogue of tachyplesin I (TP-I), CDT (KWFRVYRGIYRRR-NH(2)), in which all four cysteines were deleted. Like TP-I, CDT shows antimicrobial activity and disrupts Escherichia coli outer membrane and model membranes mimicking bacterial inner membranes at micromolar concentrations. The CDT peptide does not cause hemolysis up to 200 microg/mL while TP-I showed about 10% hemolysis at 100 microg/mL and about 25% hemolysis at 150 microg/mL. Peptide-into-lipid titrations under isothermal conditions reveal that the interaction of CDT with lipid membranes is an enthalpy-driven process. Binding assays performed using fluorometry demonstrate that the peptide CDT binds and inserts into only negatively charged membranes. The peptide-induced thermotropic phase transition of MLVs formed of DMPC and the DMPC/DMPG (7:3) mixture suggests specific lipid-peptide interactions. The circular dichroism study shows that the peptide exists as an unordered structure in an aqueous buffer and adopts a more ordered beta-structure upon binding to negatively charged membrane. The NMR data suggest that CDT binding to negatively charged bilayers induces a change in the lipid headgroup conformation with the lipid headgroup moving out of the bilayer surface toward the water phase, and therefore, a barrel stave mechanism of membrane disruption is unlikely as the peptide is located near the headgroup region of lipids. The lamellar phase (31)P chemical shift spectra observed at various concentrations of the peptide in bilayers suggest that the peptide may function neither via fragmentation of bilayers nor by promoting nonlamellar structures. NMR and fluorescence data suggest that the presence of cholesterol inhibits the peptide binding to the bilayers. These properties help to explain that cysteine residues may not contribute to antimicrobial activity and that the loss of hemolytic activity is due to lack of hydrophobicity and amphipathicity.

Anti-Infective Agents↗
PubMed2006

Redesigning the PheA domain of gramicidin synthetase leads to a new understanding of the enzyme's mechanism and selectivity.

The PheA domain of gramicidin synthetase A, a non-ribosomal peptide synthetase, selectively binds phenylalanine along with ATP and Mg2+ and catalyzes the formation of an aminoacyl adenylate. In this study, we have used a novel protein redesign algorithm, K*, to predict mutations in PheA that should exhibit improved binding for tyrosine. Interestingly, the introduction of two predicted mutations to PheA did not significantly improve KD, as measured by equilibrium fluorescence quenching. However, the mutations improved the specificity of the enzyme for tyrosine (as measured by kcat/KM), primarily driven by a 56-fold improvement in KM, although the improvement did not make tyrosine the preferred substrate over phenylalanine. Using stopped-flow fluorometry, we examined binding of different amino acid substrates to the wild-type and mutant enzymes in the pre-steady state in order to understand the improvement in KM. Through these investigations, it became evident that substrate binding to the wild-type enzyme is more complex than previously described. These experiments show that the wild-type enzyme binds phenylalanine in a kinetically selective manner; no other amino acids tested appeared to bind the enzyme in the early time frame examined (500 ms). Furthermore, experiments with PheA, phenylalanine, and ATP reveal a two-step binding process, suggesting that the PheA-ATP-phenylalanine complex may undergo a conformational change toward a catalytically relevant intermediate on the pathway to adenylation; experiments with PheA, phenylalanine, and other nucleotides exhibit only a one-step binding process. The improvement in KM for the mutant enzyme toward tyrosine, as predicted by K*, may indicate that redesigning the side-chain binding pocket allows the substrate backbone to adopt productive conformations for catalysis but that further improvements may be afforded by modeling an enzyme:ATP:substrate complex, which is capable of undergoing conformational change.

Chorismate Mutase↗
PubMed1996

Pressure-induced perturbation of apomyoglobin structure: fluorescence studies on native and acidic compact forms.

The nature of the structural changes that apomyoglobin undergoes when subjected to hydrostatic pressure, ranging from atmospheric pressure to 2.4 kbar, has been investigated by steady-state fluorescence and frequency domain fluorometry. In particular, we have examined the intrinsic tryptophanyl emission and that of the extrinsic probe 1-anilino-8-naphthalenesulfonate (ANS) bound to apomyoglobin at neutral pH, as well as at strongly acidic high-salt conditions. Apomyoglobin at neutral pH undergoes a pressure-induced structural transition, which causes the disorganization of the heme binding region with a consequent ANS dissociation; a concomitant increase in solvent accessibility to the N-terminus of the macromolecule in which tryptophans are located is also observed. At 2.4 kbar, the tryptophanyl emission is not coincident with that of a fully solvent exposed residue, thus suggesting that the N-terminal region of the apomyoglobin molecule retains elements of organized structure. The spectroscopic properties of the structural state attained at 2.4 kbar and neutral pH are different from those of the acidic compact state. The acidic compact state of apomyoglobin undergoes a pressure-induced structural change that brings the tryptophanyl residues in contact with the solvent, but does not affect the ability to bind ANS.

Acids↗
PubMed1997

Flip-flop is slow and rate limiting for the movement of long chain anthroyloxy fatty acids across lipid vesicles.

An issue that is central to understanding cellular fatty acid (FA) metabolism is whether physiologic transport of FA across cell membranes requires protein mediation or can be satisfied by the rate of spontaneous movement through the lipid phase. For this reason, considerable effort has been devoted to determining the rate-limiting steps for transport of FA across pure lipid bilayer membranes. Previously, we found that transbilayer flip-flop was the rate-limiting step for transport of long chain anthroyloxy FA (AOFA) across lipid bilayers and that the times for long chain AOFA flip-flop were > or = 100 s, yielding rate constants for flip-flop (k(ff)) that were < or = 0.01 s(-1) [Storch, J., & Kleinfeld, A. M. (1986) Biochemistry 25, 1717-1726; Kleinfeld, A. M., & Storch, J. (1993) Biochemistry 32, 2053-2061]. In those studies, k(ff) values were inferred from the time course of AOFA transfer between lipid vesicles. Recently, Kamp et al. [Kamp, F., Zakim, D., Zhang, F., Noy, N., & Hamilton, J. A. (1995) Biochemistry 34, 11928-11937], using pyranine trapped within lipid vesicles to detect flip-flop more directly, have reported that flip-flop rates of long chain AOFA are extremely rapid (k(ff) > 10 s(-1)) and are not rate limiting for transbilayer transport. Because no defect was apparent in our previous measurements, we have extended, for AOFA, the pyranine method of Kamp et al. (1995) by using stopped-flow fluorometry to resolve flip-flop rates of both short and long chain AOFA in vesicles. In addition, we have monitored the time course of transbilayer AOFA flip-flop using carboxyfluorescein (CF) trapped within the lipid vesicles as a resonance energy transfer (RET) acceptor of AO fluorescence. The differential quenching of AOFA fluorescence in the outer and inner leaflets of the bilayer allows flip-flop to be separated from the time course of AOFA binding to the vesicles. Results obtained from both the pyranine and CF methods indicate, in agreement with our previous results, that flip-flop of the long chain AOFA is slow relative to either the binding or the rate of dissociation from the vesicle. In particular, we find that the time constant (tau) for pyranine quenching by 2-AO-palmitate (2-AOPA) was > 40 s and that k(ff) obtained from RET in CF vesicles was about 0.003 s(-1). Also, in contrast to Kamp et al. (1995) who reported that k(ff) values were independent of FA chain length or structure for the C-12 to C-18 native and the C-18 AOFA within a factor of 2, we find that the rate of pyranine quenching for the shorter chain 11-AO-undecanoic acid is more than 50-fold faster than for the longer chain AOFA. We conclude, therefore, that transbilayer transport of the AOFA is limited by the rate of flip-flop and that this rate is a sensitive function of the AOFA structure.

Arylsulfonates↗
PubMed1998

Chemokine receptor-ligand interactions measured using time-resolved fluorescence.

Two G protein-coupled receptor subtypes (CXCR1 and CXCR2) mediate Interleukin-8 (IL8) action in cells. A nonradioactive lanthanide-chelate derivatized IL8 ligand was developed to measure the binding activity of the chemokine receptors, CXCR1 and CXCR2. Site-specific mutagenesis of the carboxyl-terminal serine of IL8 to cysteine resulted in a mutant IL8 (IL8-S72C) having a single free sulfhydryl. Using an iodoacetamide derivative of the Eu3+-chelate of N-(p-benzoic acid)diethylenetriamine-N,N',N"-tetraacetic acid (DTTA), incorporation of one Eu3+ per IL8 molecule ([Eu3+]IL8-S72C) was achieved. The dissociation constant for this conjugate was similar to that measured for [125I]IL8 ( approximately 2 nM) when measured by time-resolved fluorometry using CHO cell lines stably expressing CXCR1 or CXCR2 receptors. The sensitivity, stability, and high specific activity of europium-labeled IL8 demonstrate the usefulness of lanthanide-labeled proteins in the measurement of receptor-ligand interactions and may be extended to other peptide ligands.

Animals↗
PubMed1998

Quantitative analysis of the effects of intrathylakoid pH and xanthophyll cycle pigments on chlorophyll a fluorescence lifetime distributions and intensity in thylakoids.

The xanthophyll cycle-dependent dissipation of excitation energy in higher plants is one of the most important regulatory and photoprotective mechanisms in photosynthesis. Using parallel time-resolved and pulse-amplitude modulation fluorometry, we studied the influence of the intrathylakoid pH and the xanthophyll cycle carotenoids on the PSII chlorophyll (Chl) a fluorescence yield in thylakoids of Arabidopsis, spinach, and barley. Increases in concentrations of dithiothreitol in thylakoids, which have a trans-thylakoid membrane pH gradient and are known to have decreased conversion of violaxanthin (V) to zeaxanthin (Z), lead to (1) decreases in the fractional intensity of the approximately 0.5 ns Chl a fluorescence lifetime (tau) distribution component and simultaneous increases in a 1.6-1.8 ns fluorescence component and (2) increases in the maximal fluorescence intensity. These effects disappear when the pH gradient is eliminated by the addition of nigericin. To quantitatively explain these results, we present a new mathematical model that describes the simultaneous effects of the chloroplast trans-thylakoid membrane pH gradient and xanthophyll cycle pigments on the PSII Chl a fluorescence tau distributions and intensity. The model assumes that (1) there exists a specific binding site for Z (or antheraxanthin, A) among or in an inner antenna complex (primarily CP29), (2) this binding site is activated by a low intrathylakoid pH (pK approximately 4.5) that increases the affinity for Z (or A), (3) about one Z or A molecule binds to the activated site, and (4) this binding effectively "switches" the fluorescence tau distribution of the PSII unit to a state with a decreased fluorescence tau and emission intensity (a "dimmer switch" concept). This binding is suggested to cause the formation of an exciton trap with a rapid intrinsic rate constant of heat dissipation. Statistical analysis of the data yields an equilibrium association constant, Ka, that ranges from 0.7 to 3.4 per PSII for the protonated/activated binding site for Z (or A). The model explains (1) the relative fraction of the approximately 0.5 ns fluorescence component as a function of both Z and A concentration and intrathylakoid pH, (2) the dependence of the ratio of F'm/Fm on the fraction of the 0.5 ns fluorescence tau component (where F'm and Fm are maximal fluorescence intensities in the presence and the absence of a pH gradient), and (3) the dependence of the ratio of F'm/Fm on the concentration of Z and A and the intrathylakoid pH.

Carotenoids↗
PubMed1999

Modulation of flash-induced photosystem II fluorescence by events occurring at the water oxidizing complex.

The mechanism of flash-induced changes with a periodicity of four in photosystem II (PSII) fluorescence was investigated with the aim of further using fluorescence measurements as an approach to studying the structural and functional organization of the water-oxidizing complex (WOC). The decay of the flash-induced high fluorescence state of PSII was measured with pulse amplitude modulated fluorometry in thylakoids and PSII enriched membrane fragments. Calculated QA- decay was well described by three exponential decay components, reflecting QA- reoxidation with halftimes of 450 and 860 micros, 2 and 7.6 ms, and 111 and 135 ms in thylakoids and PSII membranes, respectively. The effect of modification of the PSII donor side by changing pH or by removal of the extrinsic 17 and 24 kDa proteins on period four oscillations in both maximum fluorescence yield and the relative contribution of QA- reoxidation reactions was compared to flash-induced oxygen yield. The four-step oxidation of the manganese cluster of the WOC was found to be necessary but not sufficient to produce modulation of PSII fluorescence. The capacity of the WOC to generate molecular oxygen was also required to observe a period four in the fluorescence; however, direct quenching by oxygen was not responsible for the modulation. Potential mechanisms responsible for the periodicity of four in both maximum fluorescence yield pattern and flash-dependent changes in proportion of centers with different QA- reoxidation rates are discussed with respect to intrinsic deprotonation events occurring at the WOC.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗
PubMedDate not supplied

Steam-sterilizable, fluorescence lifetime-based sensing film for dissolved carbon dioxide.

An autoclavable sensing film was developed for monitoring dissolved CO2. The sensing film, based on fluorescence resonance energy transfer (FRET), consisted of a fluorescent donor, an acceptor, and a quaternary ammonium hydroxide, which were doped in a two-component silicone film. As no aqueous solution was used in the sensing film matrix, the sensing film was unaffected by osmotic pressure. Fluorescence lifetime was selected as the sensing parameter, and measured in frequency domain using phase fluorometry. Upon exposure to 20% CO2-saturated water, a 43 degrees increase in phase angle was observed at 100 MHz. The process was fully reversible when the sensing film was exposed to nitrogen-saturated water. The estimated response and recovery times for 90% signal change were 1 min (for a step change from 0 to 6.7% CO2-saturated water) and 1.5 min (for a step change from 6.7 to 3.3% CO2-saturated water). When used for on-line monitoring of dissolved CO2 produced by a culture of Escherichia coli, the sensing film showed a similar trend to that obtained from off-line measurements using a wet chemistry analyzer.

Carbon Dioxide↗
PubMed2004

Measurement of mixing-controlled reactive transport in homogeneous porous media and its prediction from conservative tracer test data.

Numerical and theoretical studies have indicated that pore-scale mixing can be the limiting process for reactions among dissolved compounds in porous media. It has been claimed that multicomponent reactions in porous media could be accurately estimated using mixing coefficients obtained from point-like measurements of conservative tracer concentrations. In this study, we verify these concepts experimentally by tracer tests in a homogeneously packed saturated sand column. Fiber-optic fluorometry was applied to detect point-related concentrations of fluorescein, which was used as both the conservative and the reactive tracer. In the reactive tracer experiment, an acidic solution containing the tracer was displaced by an alkaline solution without tracer. Since the fluorescence of fluorescein is quenched at low pH, the fluorescence intensity measured in the reactive breakthrough curve indicated the mixing of the two solutions. The measured reactive breakthrough curves were compared to predictions based on the conservative breakthrough curves. Predictions and measurements agreed well. Our results imply that incomplete mixing on the pore scale is of minor significance for field-scale applications. On this scale, however, even weak sorption might influence mixing significantly.

Contrast Media↗
PubMed2002

A fluorescence-based method for direct measurement of submicrosecond intramolecular contact formation in biopolymers: an exploratory study with polypeptides.

A fluorescent amino acid derivative (Fmoc-DBO) has been synthesized, which contains 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) as a small, hydrophilic fluorophore with an extremely long fluorescence lifetime (325 ns in H2O and 505 ns in D2O under air). Polypeptides containing both the DBO residue and an efficient fluorescence quencher allow the measurement of rate constants for intramolecular end-to-end contact formation. Bimolecular quenching experiments indicated that Trp, Cys, Met, and Tyr are efficient quenchers of DBO (k(q) = 20, 5.1, 4.5, and 3.6 x 10(8) M(-1) x s(-1) in D2O), while the other amino acids are inefficient. The quenching by Trp, which was selected as an intrinsic quencher, is presumed to involve exciplex-induced deactivation. Flexible, structureless polypeptides, Trp-(Gly-Ser)n-DBO-NH2, were prepared by standard solid-phase synthesis, and the rates of contact formation were measured through the intramolecular fluorescence quenching of DBO by Trp with time-correlated single-photon counting, laser flash photolysis, and steady-state fluorometry. Rate constants of 4.1, 6.8, 4.9, 3.1, 2.0, and 1.1 x 10(7) s(-1) for n = 0, 1, 2, 4, 6, and 10 were obtained. Noteworthy was the relatively slow quenching for the shortest peptide (n = 0). The kinetic data are in agreement with recent transient absorption studies of triplet probes for related peptides, but the rate constants are significantly larger. In contrast to the flexible structureless Gly-Ser polypeptides, the polyproline Trp-Pro4-DBO-NH2 showed insignificant fluorescence quenching, suggesting that a high polypeptide flexibility and the possibility of probe-quencher contact is essential to induce quenching. Advantages of the new fluorescence-based method for measuring contact formation rates in biopolymers include high accuracy, fast time range (100 ps-1 micros), and the possibility to perform measurements in water under air.

Amino Acids↗