Ca2+ binding kinetics of fura-2 and azo-1 from temperature-jump relaxation measurements.

The Ca2+-binding kinetics of fura-2 and azo-1 were studied using temperature-jump relaxation methods. In 140 mM KCl at 20 degrees C, the association and dissociation rate constants for fura-2 were 6.02 x 10(8) M-1s-1 and 96.7 s-1, respectively. The fura-2 kinetics were insensitive to pH over the range 7.4 to 8.4. Azo-1 was studied in 140 mM KCl, at pH 7.4, at 10 degrees and 20 degrees C. At 10 degrees C, azo-1 exhibited association and dissociation rate constants of 1.43 x 10(8) M-1s-1 and 777.9 s-1, respectively; while at 20 degrees C, the corresponding values were 3.99 x 10(8) M-1s-1 and 1,177 s-1. The kinetic results demonstrate that fura-2 and azo-1 are well suited to monitoring rapid changes in intracellular [Ca2+].     

Investigation of noncalcium interactions of fura-2 by classical and synchronous fluorescence spectroscopy.

Several authors have reported unexpected intracellular spectra of both indo-1 and fura-2. One of the major methodological problems in the evaluation of calcium concentration using fluorescent probes is that it is assumed that only two forms of the dyes are detectable within the cells. We show in this study of fura-2 properties that this calcium probe is pH-sensitive and able to bind to cellular proteins. The excitation spectra of protonated and protein-bound forms of fura-2 exhibit a maximum in the same region as that associated with the calcium-free form (i.e., near 365 nm). The very small shift in the excitation spectra upon proton or protein binding precludes the use of classical methods to determine the spectral composition of mixtures of several forms of fura-2. We therefore used the synchronous fluorescence technique to detect the protein-bound form of fura-2 selectively, in order to assess the pH dependence of the fura-2/protein interaction. The nonspecific binding of fura-2 to proteins is reinforced at acidic pH and inhibited by calcium. The fact that the same type of interaction was found between fura-2 and poly-L-lysine suggests that it could be mediated by basic amino acids. Because of the strong overlap of the excitation spectrum of the unprotonated free fura-2 with those associated with the protonated and protein-bound forms, a cytoplasmic acidification may lead to an artifactual measurement of low calcium levels.     

Interpreting photoactivated fluorescence microscopy measurements of steady-state actin dynamics.

A continuum model describing the steady-state actin dynamics of the cytoskeleton of living cells has been developed to aid in the interpretation of photoactivated fluorescence experiments. In a simplified cell geometry, the model assumes uniform concentrations of cytosolic and cytoskeletal actin throughout the cell and no net growth of either pool. The spatiotemporal evolution of the fluorescent actin population is described by a system of two coupled linear partial-differential equations. An analytical solution is found using a Fourier-Laplace transform and important limiting cases relevant to the design of experiments are discussed. The results demonstrate that, despite being a complex function of the parameters, the fluorescence decay in photoactivated fluorescence experiments has a biphasic behavior featuring a short-term decay controlled by monomer diffusion and a long-term decay governed by the monomer exchange rate between the polymerized and unpolymerized actin pools. This biphasic behavior suggests a convenient mechanism for extracting the parameters governing the fluorescence decay from data records. These parameters include the actin monomer diffusion coefficient, filament turnover rate, and ratio of polymerized to unpolymerized actin.     

Quantitation of intracellular free calcium in single adult cardiomyocytes by fura-2 fluorescence microscopy: calibration of fura-2 ratios

Isolated rat myocytes incubated with the acetoxy methyl ester of fura 2 contained partially hydrolyzed esters, necessitating in vivo calibration of the signals obtained by fluorescence microscopy for calculation of pCa. Ionophores did not produce reliable R'max and R'min values in respiring myocytes, and elevated free calcium caused individual cells to hypercontract and burst. These difficulties were overcome by superfusion with a glucose-free buffer containing an inhibitor and an uncoupler of oxidative phosphorylation. R'max and R'min values obtained by ionophore treatment of deenergized myocytes were normalized to an in vitro calibration curve. Resting pCa derived from the individual curves averaged 6.9 for calcium-tolerant rod-shaped myocytes.     

Fatty acid-enhanced binding of flavin mononucleotide to bacterial luciferase measured by steady-state fluorescence.

Bacterial luciferase catalyzes the oxidation of reduced flavin mononucleotide and tetradecanal resulting in the emission of light. We have investigated the interactions of a recombinant luciferase from a terrestrial bacterium Xenorhabdus luminescens with the reaction products, FMN and myristic acid, using steady-state fluorescence spectroscopy. Quenching of the intrinsic fluorescence and FMN fluorescence on binding of FMN to luciferase was found to be greatly stimulated in the presence of myristic acid, corresponding to a reduction of more than 30-fold in the FMN dissociation constant of the enzyme. In addition, the FMN-luciferase complex exhibits distinct fatty acid-dependent fluorescence properties. These results indicate that luciferase forms a ternary complex with FMN and myristic acid with a significantly different conformation from that of the binary FMN-luciferase complex.     

Visualization and measurement of calcium transients in Amoeba proteus by fura-2 fluorescence

A fura-2 microspectrofluorimeter was used to visualize and measure intracellular calcium transients in normal locomoting and experimentally treated Amoeba proteus. The results show that subcellular heterogeneities of cytosolic free calcium, [Ca2+]i, correlate in time and distribution with characteristic patterns of protoplasmic streaming and ameboid movement. In detail, calcium ions have a dual effect by regulating both the contractile activities of the actomyosin cortex and the rheological properties of the cytoplasmic matrix. A high resting [Ca2+]i of 1.5 to 2.0 x 10(-7) M in the uroid region or in retracting pseudopodia is associated with the transformation of rigid ectoplasmic gel into fluid endoplasmic sol, and a low [Ca2+]i of 10(-9) to 10(-8) M in the front region or in extending pseudopodia with the re-transformation of endoplasmic sol into ectoplasmic gel. Locally increased peripheral [Ca2+]i accumulations higher than 10(-7) M are also observed at places where the actomyosin cortex is known to generate motive force by contraction, i.e., in the intermediate region of orthotactic amebas or in large pseudopodia of polytactic cells. External application of 30 mM KCl abolishes the intracellular Ca2+ gradient such that [Ca2+]i attains a uniform distribution and a maximum concentration of 2 x 10(-7) M; as a consequence, cells can show a transient loss of their locomotor activity and polarity by undergoing spherulation and total contraction. On the other hand, high external Ca2+ concentrations in the range of 100 mM stabilize the bipolar cellular organization, enhance the movement velocity and induce the propagation of Ca2+ waves repeatedly running from the uroid to the front region. The significance of external ions for signal transmission and the control of dynamic activities as well as the origin and fate of calcium participating in the observed transients are discussed.     

Nanosecond fluorescence microscopy. Emission kinetics of fura-2 in single cells.

A microscope based time-correlated single photon counting instrument has been constructed to measure fluorescence intensity and emission anisotropy decays from fluorophores in single cells on a nanosecond time scale. The sample is excited and the emission collected using epi-illumination optics with frequency-doubled pulses from the cavity-dumped output of a synchronously pumped dye laser serving as an excitation source. Collection of decays from a single cell is possible due to the presence of an iris in the emission path that can be reduced to less than the diameter of a single cell. Using the instrument the decay of 60 nM 1,6-diphenyl-1,3,5-hexatriene was measured, demonstrating that adequate data for lifetime analysis can be recorded from fewer 10(3) molecules of the fluorophore in an illuminated volume of 23 fl. In addition, the intensity and anisotropy decays of fura-2 in single adherent cells and in suspensions of fura-2 loaded cells in suspension, although the relative amplitudes and decay constants vary somewhat from cell to cell. The results indicate that a significant but variable fraction of fura-2 is bound to relatively immobile macromolecular components in these cells.     

Chlorpheniramine binding to human serum albumin by fluorescence quenching measurements.

The binding of chlorpheniramine to human serum albumin has been studied by fluorescence quenching, as a function of temperature; the experimental data could only be fitted to the Stern-Volmer modified equation. A statistical analysis of the results was performed in order to determine the significance of the constants calculated by this equation, as well as their thermodynamic parameters. The chlorpheniramine binding to human serum albumin accounts for almost half of the binding of this antihistaminic agent to human plasma proteins.     

Calcium measurements in perfused mouse heart: quantitating fluorescence and absorbance of Rhod-2 by application of photon migration theory

Both theoretical and experimental results are presented for the quantitative detection of calcium transients in the perfused mouse heart loaded with the calcium-sensitive fluorescent dye Rhod-2. Analytical models are proposed to calculate both the reflected absorbance and fluorescence spectra detected from the mouse heart. These models allow correlation of the measured spectral intensities with the relative quantity of Rhod-2 in the heart and measurement of the changes in quantum yield of Rhod-2 upon binding calcium in the heart in which multiple scattering effects are predominant. Theoretical modeling and experimental results demonstrate that both reflected absorbance and fluorescence emission are attenuated linearly with Rhod-2 washout. According to this relation, a ratiometric method using fluorescence and absorbance is validated as a measure of the quantum yield of calcium-dependent fluorescence, enabling determination of the dynamics of cytosolic calcium in the perfused mouse heart. The feasibility of this approach is confirmed by experiments quantifying calcium transients in the perfused mouse heart stimulated at 8 Hz. The calculated cytosolic calcium concentrations are 368 +/- 68 nM and 654 +/- 164 nM in diastole and systole, respectively. Spectral distortions induced by tissue scattering and absorption and errors induced by the geometry of the detection optics in the calcium quantification are shown to be eliminated by using the ratio method. Methods to effectively minimize motion-induced artifacts and to monitor the oxygenation status of the whole perfused heart are also discussed.     

Endogenous heavy metal ions perturb fura-2 measurements of basal and hormone-evoked Ca2+ signals.

Using the membrane-permeant chelator of heavy metal ions, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylene diamine (TPEN), we demonstrate that in pancreatic acinar cells, hepatocytes, and a variety of mammalian cell lines, endogenous heavy metal ions bind to cytosolic fura-2 causing basal cytosolic free [Ca2+] ([Ca2+]i) to be overestimated. TPEN had most effect in cells lightly loaded with fura-2, suggesting the presence of a limited pool of heavy metal ions (> or = 12 microM in pancreatic acinar cells) that does not rapidly exchange across the plasma membrane. In fura-2-loaded hepatocytes, vasopressin failed to evoke a detectable change in fluorescence, but after preincubation of cells with TPEN, it caused fluorescence changes characteristic of an increase in [Ca2+]i. We conclude that in many mammalian cells, a slowly exchanging mixture of cytosolic heavy metal ions binds to fura-2 both to quench its fluorescence and to mimic the effects of Ca2+ binding, thereby causing basal [Ca2+]i to be overestimated. By chelating endogenous heavy metal ions, TPEN allows basal [Ca2+]i to be accurately measured and, by preventing competition between heavy metal ions and Ca2+ for binding to fura-2, unmasks the full effect of agonists in increasing [Ca2+]i.     

Temperature response and photoinhibition investigated by chlorophyll fluorescence measurements for four distinct species of dipterocarp trees

The effects of strong light in combination with elevated temperatures on the photosynthetic system were examined in 4 dipterocarp tree species with ecologically different habitats. The 4 dipterocarp tree species were: Shorea platyclados originated from upper dipterocarp forests, Shorea parvifolia– lowland and hill dipterocarp forests, Shorea assamica– lowland dipterocarp forests, and Dipterocarpus oblongifolius– riparian fringes. S. platyclados and D. oblongifolius have higher growth and survival rates in open sites than S. parvifolia and S. assamica. Tolerance of high temperature among the species was assessed by determining the critical temperatures (T_c) at which the minimal fluorescence (F_o) began to rise sharply. This was measured by exposing plants to an increasing temperature of about 1°C min^?1. The intrinsic thermotolerance of the thylakoid membrane appears to be the highest for D. oblongifolius (T_c=46.4°C), intermediate for S. platyclados (45.7°C), and lowest for S. parvifolia and S. assamica (45.2 and 45.3°C, respectively). The temperature‐dependent efficiency of PSII electron transport (ΔF/F′_m), photochemical quenching (q_P), and the efficiency of light capture of open PSII (F′_v/F′_m) were measured at the photosynthetic steady state at least 10 min after the light exposure (180 μmol m^?2 s^?1 PFD). Stable temperature responses of ΔF/F′_m and q_P were observed in S. platyclados and D. oblongifolius, while those in S. parvifolia and S. assamica were more temperature‐dependent and severely affected at 45°C. Little difference was observed in temperature‐dependent F′_v/F′_m among species. Photoinhibitory light exposure (1600 μmol m^?2 s^?1 PFD) for 2 h at 40°C had little effect on the recovery kinetics from photoinhibition of S. platyclados and D. oblongifolius compared with those at 35°C. In contrast, the recovery from photoinhibition was retarded in S. parvifolia and S. assamica. These findings suggest that even at 40°C, a temperature below T_c, an exposure to strong light exacerbated photoinhibition in S. parvifolia and S. assamica corresponding to the closure of PSII reaction centers, as indicated by the decrease in q_P at this temperature. Thus, S. platyclados and D. oblongifolius, which occur at uplands and riparian fringes with frequent disturbances, are suggested to have higher photosynthetic tolerance to elevated temperatures contributing to a circumvention of photoinhibition.     

Nucleotide flipping by restriction enzymes analyzed by 2-aminopurine steady-state fluorescence

Many DNA modification and repair enzymes require access to DNA bases and therefore flip nucleotides. Restriction endonucleases (REases) hydrolyze the phosphodiester backbone within or in the vicinity of the target recognition site and do not require base extrusion for the sequence readout and catalysis. Therefore, the observation of extrahelical nucleotides in a co-crystal of REase Ecl18kI with the cognate sequence, CCNGG, was unexpected. It turned out that Ecl18kI reads directly only the CCGG sequence and skips the unspecified N nucleotides, flipping them out from the helix. Sequence and structure conservation predict nucleotide flipping also for the complexes of PspGI and EcoRII with their target DNAs (/CCWGG), but data in solution are limited and indirect. Here, we demonstrate that Ecl18kI, the C-terminal domain of EcoRII (EcoRII-C) and PspGI enhance the fluorescence of 2-aminopurines (2-AP) placed at the centers of their recognition sequences. The fluorescence increase is largest for PspGI, intermediate for EcoRII-C and smallest for Ecl18kI, probably reflecting the differences in the hydrophobicity of the binding pockets within the protein. Omitting divalent metal cations and mutation of the binding pocket tryptophan to alanine strongly increase the 2-AP signal in the Ecl18kI–DNA complex. Together, our data provide the first direct evidence that Ecl18kI, EcoRII-C and PspGI flip nucleotides in solution.     

Intracellular pH-determination by fluorescence measurements.

A method was developed to determine the intracellular pH (pHi) of individual cells by use of fluorescence measurements. The method is based on the observation that the fluorescence excitation spectrum of fluorescein is pH-dependent. Fluorescence excitation spectra from individual rat bone marrow cells treated with fluorescein diacetate (FDA) were compared with those of fluorescein solutions of known pH values. Cells which were suspended in media of pH between 4.0 and 8.1 with high to normal buffering capacities had pHi values equal to those of the media. Cells suspended in media with low buffering capacities maintained a pH,i of 6.7 +/- 0.2. Preliminary results indicated that the pHi of individual cells may also be determined by using flow cytometry.     

Direct fluorescence measurements of Mg2+ binding to sarcoplasmic reticulum ATPase

In the absence of calcium, interaction of magnesium with SR-ATPase induced a blue shift in intrinsic fluorescence emission. This Mg2+-induced fluorescence change was pH-dependent and an apparent Mg dissociation constant of 5 mM was found at pH 7. Equilibrium studies showed that magnesium competes for the high affinity Ca2+ binding sites and stopped flow measurements of the transient kinetics indicated a multistep interaction between magnesium and the calcium pump. These results suggest that magnesium drives the sarcoplasmic reticulum atpase toward an E.Mg species which might be a dead-end complex.     

Perturbation of sarcoplasmic reticulum calcium release and phenol red absorbance transients by large concentrations of fura-2 injected into frog skeletal muscle fibers

Intact single twitch fibers from frog muscle were studied on an optical bench apparatus after micro-injection with two indicator dyes: phenol red, to monitor a previously described signal (denoted delta pHapp; Hollingworth and Baylor. 1990. J. Gen. Physiol. 96:473-491) possibly reflective of a myoplasmic pH change following action potential stimulation; and fura-2, to monitor the associated change in the myoplasmic free calcium concentration (delta[Ca2+]). Additionally, it was expected that large myoplasmic concentrations of fura-2 (0.5-1.5 mM) might alter delta pHapp, since it was previously found (Baylor and Hollingworth. 1988. J. Physiol. 403:151-192) that the Ca2(+)-buffering effects of large fura-2 concentrations: (a) increase the estimated total concentration of Ca2+ (denoted by delta[CaT]) released from the sarcoplasmic reticulum (SR), but (b) reduce and abbreviate delta[Ca2+]. The experiments show that delta pHapp was increased at the larger fura-2 concentrations; moreover, the increase in delta pHapp was approximately in proportion to the increase in delta[CaT]. At all fura-2 concentrations, the time course of delta pHapp, through time to peak, was closely similar to, although probably slightly slower than, that of delta[CaT]. These properties of delta pHapp are consistent with an hypothesis proposed by Meissner and Young (1980. J. Biol. Chem. 255:6814-6819) and Somlyo et al. (1981. J. Cell Biol. 90:577-594) that a proton flux from the myoplasm into the SR supplies a portion of the electrical charge balance required as Ca2+ is released from the SR into the myoplasm. A comparison of the amplitude of delta pHapp with that of delta[CaT] indicates that, in response to a single action potential, 10-15% of the charge balance required for Ca2+ release may be carried by protons.     

Microtubule treadmilling in vitro investigated by fluorescence speckle and confocal microscopy.

Whether polarized treadmilling is an intrinsic property of microtubules assembled from pure tubulin has been controversial. We have tested this possibility by imaging the polymerization dynamics of individual microtubules in samples assembled to steady-state in vitro from porcine brain tubulin, using a 2% glycerol buffer to reduce dynamic instability. Fluorescence speckled microtubules were bound to the cover-glass surface by kinesin motors, and the assembly dynamics of plus and minus ends were recorded with a spinning-disk confocal fluorescence microscopy system. At steady-state assembly, 19% of the observed microtubules (n = 89) achieved treadmilling in a plus-to-minus direction, 34% in a minus-to-plus direction, 37% grew at both ends, and 10% just shortened. For the population of measured microtubules, the distribution of lengths remained unchanged while a 20% loss of original and 27% gain of new polymer occurred over the 20-min period of observation. The lack of polarity in the observed treadmilling indicates that stochastic differences in dynamic instability between plus and minus ends are responsible for polymer turnover at steady-state assembly, not unidirectional treadmilling. A Monte Carlo simulation of plus and minus end dynamics using measured dynamic instability parameters reproduces our experimental results and the amount of steady-state polymer turnover reported by previous biochemical assays.     

Electrostatic surface properties of plasmalemma vesicles from oat and wheat roots. Ion binding and screening investigated by 9-aminoacridine fluorescence

Right-side-out and sealed plasmalemma vesicles were isolated from roots of spring wheat (Triticum aestivum L. cv. Drabant) and oat (Avena sativa L. cv. Brighton) by two-phase partition in a medium containing sucrose (0.25 mol l^-1). Oat root plasmalemma vesicles were discovered to contain a strongly fluorescent compound with an emission maximum at 418 nm. The surface potential of the membranes was monitored by 9-aminoacridine fluorescence and the effect of protein concentration, mannitol versus sucrose, absence of osmoticum, concentrations of salt, and titrations with chelators investigated. It is concluded that i) protein concentrations of less than 50 g ml^-1 for oat and 100 g ml^-1 for wheat plasmalemma vesicles should be used to avoid serious problems with non-linearity of response of 9-aminoacridine fluorescence, ii) mannitol can be used instead of sucrose as the osmoticum, iii) the vesicles were ruptured in the absence of osmoticum allowing us to monitor both sides of the membranes, iv) plasmalemma vesicles from oat roots are more negative than vesicles from wheat roots, and v) oat and wheat root plasmalemma vesicles are isolated with about the same amounts of bound Ca²⁺ and Mg²⁺. These bound divalent cations may not, however, reflect the in-vivo conditions since the tissues were homogenised in the presence of ethylenediaminetetraacetic acid.Abbreviations EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - c1/2 value concentration at which half of the maximum effect is observed - Mops 3-(N-morpholino)propanesulfonic acid     

The primary event in vision investigated by time-resolved fluorescence spectroscopy.

The picosecond fluorescence kinetics and quantum yield from bovine rhodopsin were measured in the 5-40 degrees K range. The fluorescence rise and decay times are faster than our resolution of 15 ps (full width at half maximum) over this entire temperature range. The size of the observed emission was also temperature independent, and we find that the upper limit of rhodopsin's fluorescence quantum yield to be phi f approximately equal to 10(-5). Replacing all of rhodopsin's exchangeable protons with deuterons by suspending rhodopsin in D2O had no effect on either the kinetics of the emission or the value of the quantum yield. Our data provide strong confirmation of the idea that the first step in the visual process is an excited-state cis-to-trans isomerization about the C11-C12 double bond of retinal.     

Investigation of conformational changes in yeast enolase using dynamic fluorescence and steady-state quenching measurements

Conformational changes in yeast enolase were investigated using steady state quenching and dynamic (fluorescence decay and fluorescence anisotropy decay) measurements. The tryptophan fluorescence rotational correlation time increases from 24 to 38 ns on subunit association. The acrylamide quenching constant decreases two-fold when the subunits associate. The conformational metal ion effect suggests a more compact molecule. Under conditions of catalysis, the correlation time decreases 25%, though the sedimentation constant does not change (Holleman, 1973). The enzyme may undergo a hinge-bending motion during catalysis.     

Binding of beta-naphthyl triphosphate to human adult hemoglobin accompanying deoxygenation. Investigated by simultaneous measurements of fluorescence, absorbance and partial pressure of oxygen

Binding of a fluorescent allosteric effector, beta-naphthyl triphosphate (beta-NapP3), to human adult hemoglobin (HbA) at various levels of oxygen saturation were investigated by simultaneous measurements of fluorescence, absorbance and oxygen partial pressure. Amounts of beta-NapP3 bound to HbA were easily estimated from the fluorescence intensities of HbA solutions, because it was previously proved that the fluorescence of beta-NapP3 bound to HbA is completely quenched. Exchange reactions of the above fluorescent allosteric effector with 2,3-bisphosphoglycerate (DPG) were also examined at various levels of oxygen saturation. It was found that beta-NapP3 binds to deoxyHbA tetramer in the molar ratio of 2:1, and that one of the two beta-NapP3 competes with DPG. It was also found that beta-NapP3 binds to completely oxygenated HbA tetramer in the molar ratio of 1:1, and that the bound beta-NapP3 was not released by adding DPG. The binding affinity of beta-NapP3 for the noncompetitive site of completely oxygenated HbA, to which DPG does not bind, was smaller than that for the noncompetitive site of deoxyHbA, to which DPG also does not bind. Furthermore, the correlations between oxygen bindings by HbA and the bindings of beta-NapP3 to HbA in the intermediate stages of deoxygenation were investigated. It was revealed that HbA as a tetramer exists in three conformational states rather than simple two states as Monod, Wyman, and Changeux had proposed.