Jean Vigo

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.

Native Fluorescence and Mag-Indo-1-Protein Interaction as Tools for Probing Unfolding and Refolding Sequences of the Bovine Serum Albumin Subdomain in the Presence of Guanidine Hydrochloride

Changes in the fluorescence spectrum of tryptophans Trp 134 and Trp 212 in bovine serum albumin (BSA) and of Trp 214 of human serum albumin in the presence of the chaotropic agent guanidine hydrochloride (Gnd) were studied. A detailed analysis of the fluorescence spectrum of native BSA yielded the fluorescence spectrum for each tryptophan of BSA. Modifications in the binding of Mag-indo-1 to BSA, which results in a specific quenching of the fluorescence spectrum of Trp 134 associated with an energy transfer from Trp 134 to the protein-bound Mag-indo-1, were also investigated. Changes occurring when the Gnd concentration is decreased stepwise cover a larger concentration scale of Gnd than the reverse protocol, allowing one to suggest that the resulting conformational changes in the subdomain IA of BSA involve at least three different steps.

Microspectrofluorometry as a tool for investigation of non-calcium interactions of Indo-1

Indo-1 is a fluorescent calcium probe used to measure intracellular free calcium concentrations. These measurements are often performed by comparing the fluorescence intensities of Indo-1-treated cells at two selected wavelengths corresponding to the maxima of the fluorescence spectra of the calcium-bound and calcium-free forms. In this study, we used an optical multichannel analyser to numerise the fluorescence emitted by a single cell. A computerised resolution of numerised spectra was used on intracellular Indo-1 fluorescence. Calculation of numerical and graphic estimators allows us to evaluate the fit of the resolution. Different sets of characteristic spectra were compared using this method. It appeared that no linear combination of the two known forms of Indo-1 and of the cell autofluorescence can fit with spectra of Indo-1-treated cells. In addition, a study of the physico-chemical properties of Indo-1 shows the existence of two other forms of the molecule: a protonated form (maximum emission at 455 nm) and a form in interaction with proteins (maximum emission at 438 nm). Taking into account the contribution of these two new forms leads to an improved spectral resolution of the fluorescence of Indo-1-treated living cells and, therefore, improves calcium measurements. Moreover, quantification of the amount of the protonated form of Indo-1 allows a measurement of intracellular pH at the same time as calcium determination.

Interaction of a protein, BSA, and a fluorescent probe, Mag-Indo-1, influence of EDTA and calcium on the equilibrium.

Recent findings indicate that ion-chelator probes with tetracarboxylate structure bind proteins. It was suggested that these fluorescent probes are valuable tools to gain information on protein structure through the energy transfer from tryptophans to the bound probe. Here, the binding of the fluorescent probe Mag-Indo-1 to bovine serum albumin (BSA) was investigated. Mag-Indo-1 was reported previously to serve as a probe for magnesium cations (Kd = 2.8 x 10(-4) M for zero ionic strength) which can also interact with calcium cations (Kd = 7.5 x 10(-7) M). Probe complexation with protein results in a shift of the emission fluorescence spectrum of the probe from 480 to 457 nm. We used emission fluorescence techniques to monitor this interaction. Computational resolution of the complex fluorescence spectra and a new software to test the theoretical model were developed in our laboratory. This enabled us to calculate the number of interacting sites and the dissociation constants. The fluorescent probe Mag-Indo-1 binds at a singular site with high affinity (Kd = 1.8 x 10(-7) M) to bovine serum albumin (BSA). Since proteins are known to bind several compounds unspecifically, we have studied the influence of EDTA as a competitor of the probe. Our findings suggest that the BSA binding site is identical for both Mag-Indo-1 and EDTA. We found that EDTA binds the protein with Kd = 0.4 x 10(-3) M. We studied the influence of calcium and found that Mag-Indo-1 does not bind the calcium free Apo-protein anymore.

Lifetime of Fluorescent Pyrene Butyric Acid Probe in Single Living Cells for Measurement of Oxygen Fluctuation

We study the fluorescence lifetime of the well‐known 1‐pyrene butyric acid (PBA) to assess oxygen concentrations in living cells. The behavior of the probe is first studied in water, ethanol, protein solution and liposome suspension. The Stern‐Volmer plot of these solutions is linear, and the bimolecular reaction rate constant agrees with previous observations. In single living cells, the PBA lifetime decreases with oxygen concentration (185 to 55 ns). The probe lifetime differences between living cells and liposome suspension, especially under nitrogen atmosphere, suggest a supplemental pathway for the deactivation of the probe. We simplify further the complex living cells system by stopping the cell functions and studying freshly fixed cells. In this case, we obtained an increase of PBA lifetime under nitrogen atmosphere (215 ns).

C-SNARF-1 as a pHi fluoroprobe: discrepancies between conventional and intracellular data do not result from protein interactions

Abstract Although the fluorescent probe C-SNARF-1 has been found useful for intracellular pH evaluation, practical problems have been reported. Some of these problems have been tentatively related to interactions between C-SNARF-1 and intracellular proteins. Nevertheless, such interactions have yet to be reported. Using an efficient method of resolution of fluorescence spectra, a careful analysis of the respective chemical properties of the commercially available C-SNARF-1 and of the product of the enzymatic hydrolysis of C-SNARF-1/AM has been performed. Our data demonstrate that no interaction occurs between the last compound and proteins such as bovine serum albumin (BSA). On the contrary a contaminant able to bind with BSA was found in the sample of the commercially available C-SNARF-1. Such findings may explain why some studies have had difficulty correlating their intracellular data with those obtained in living cells. They also exemplify the advantage of using a more sophisticated method than the usual ratiometric one to interprete intracellular fluorescence spectra.

Are intracellular ionic concentrations accessible using fluorescent probes? The example of Mag-indo-1.

The study of the physicochemical properties of Mag-indo-1, a fluorescent probe used for intracellular magnesium measurements, has shown that in a biological environment the deprotonated form of this probe is in simultaneous equilibrium with a protonated form, a protein and a magnesium-bound form. The complex emission fluorescence spectrum emitted by a single living cell was analyzed using a computerized method, allowing the evaluation of the Mag-indo-1 to the cellular fluorescence. This approach used to evaluate intracellular Mg2+ concentration has also shown the variability of the important participation of protein-bound Mag-indo-1 to the cellular fluorescence. Thus the widely used ratioing method, unable to take into account this variability, cannot afford a reliable evaluation of [Mg2+]. Whatever the technique used for investigation (microfluorimetry, flow cytometry, etc.) the evaluation of [Mg2+]i using the fluorescent probe Mag-indo-1 requires a method able to quantify, in complex fluorescence, the fluorescence intensity of the forms involved in the equilibrium with Mg2+.

Quantitative microfluorometry of isolated living cells with pulsed excitation: Development of an effective and relatively inexpensive instrument

Using a computer interfaced original apparatus, we can analyze the fluorescence decay time of a single living cell. This apparatus can measure rate constants in the range of one to few hundred nanoseconds. The smallest sample analyzed was 5×5×5 μm. We obtained enough sensitivity to measure the fluorescence of about 105 molecules of benzo(a)pyrene with a signal/noise ratio equal to 5. The signal was then defined with 300 points and experiment was 60 s long with a laser pulse frequency equal to 100 Hz. Distortions were less than 3% of the signal. Different kinds of information became accessible: statistical comparison on cellular populations and variation with time of studied parameters. As an example of applications, the intrinsic cell fluorescence has been analyzed in order to determine the amounts of bound and free NAD(P)H in single living cells.

Adriamycin dose and time effects on cell cycle, cell death, and reactive oxygen species generation in leukaemia cells.

We investigate the relative importance of the different mechanisms of Adriamycin, an anthracycline, and their interrelations, in particular the link between cell cycle arrest, cell death, and generation of reactive oxygen species (ROS) that is suspected to be the origin of cardiotoxic side-effects. We introduced a lifetime fluorescence based technology and used videomicrofluorometry, two efficient analytical methods. We show that depending on the doses and time after incubation, ADR will not reach the same compartments (nucleus, mitochondria, cytosol) in the cells, having consequences on the production of ROS, growth arrest pathways and cell death pathways.

Acid—base and calcium-binding properties of the fluorescent calcium indicator indo-1

Abstract Indo-1, a tetracarboxylate fluorescent probe, is commonly used for monitoring intracellular calcium levels. Indo-1 is calibrated at pH > 7, whereas intracellular pH is, in many cases, below pH 7. Therefore, the behaviour of the probe was studied at these pH values. We present the acid—base and calcium-binding properties of the molecule, as revealed by absorption and fluorescence spectroscopy, within the pH range 3 – 8. Two protonated forms of Indo-1 are described. The corresponding acidity constants are determined ( i.e. p K a 1 = 6.2, p K a 2 = 4.35). The existence of a protonated form in the physiological domain could seriously limit quantitative applications of the ‘ratio’ method, which is generally used for intracellular measurements. An improved physicochemical model is proposed, and the three-dimensional structures of the single protonated and calcium-bound forms of Indo-1 are given.