Jean-Marie Salmon

MICROSPECTROFLUOROMETRIC APPROACH TO THE STUDY OF FREE/BOUND NAD(P)H RATIO AS METABOLIC INDICATOR IN VARIOUS CELL TYPES

Under excitation at 365 nm, the cell fluorescence is mainly due to bound and free NAD(P)H, plus a small contribution from flavins. Resolution is first attempted in the simplest case. i.e. the increase spectrum (δIf) due to microinjection of glucose‐6‐phosphate (G6P) into EL2 ascites cells. Above 510 nm, δIF is identical to the spectrum of free NADH. Below 510 nm. the presence of a second component is suggested, i.e. the intensity of the free NADH spectrum is lower than the measured δIF level. The difference between δIf and the free NADH spectrum (maximum at 475 nm) yields a spectrum suggestive of bound NADH with maximum at 450 nm. Thus, with free and bound NADH, the entire δIF can be reconstructed, with some assumptions as to the relative quantum yields of the two components. This seems to leave no place for a flavin component.

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.

A microspectrofluorometric approach for the study of Benzo(a)pyrene and Dibenzo(a, h)anthracene metabolization in single living cells

SummaryThe use of a microspectrofluorometer in conjunction with the microelectrophoretic intracellular injection of glycolytic intermediates, for the study of carcinogen metabolization (i.e. Benzo(a)pyrene (BP) and Dibenzo(a, h)anthracene) by single living EL2 ascites cells, has allowed the detection of fluorescence attributable to the metabolites of BP and DBA.The results obtained are in agreement with those yielded by more conventional methods: i.e. in vivo assay and in vitro reconstitution of intracellular environment. Thus, it is observed that NADPH is more active in the metabolization of BP. Furthermore, in the case of BP the fluorescence attributable to a BP metabolite, exhibits a strong similarity to 3-OH benzo(a) pyrene.

32‐msec scan of the NAD(P)H fluorescence spectrum in single living cells

A multichannel microspectrofluorometer based on an electron bombardment silicon camera tube, a multiscaling computer, and a miniature Amici prism for spectrometer, allows the scanning of the NAD(P)H fluorescence emission spectrum from a 30 μ region in a single living EL2 ascites cancer cell, within 32 msec. Observations of changes in the relative maximum emission intensities of two fluorochromes within a same cell are quite possible (for emission maxima ∼ 10–20 nm apart). Thus, in the fluorescence emission spectrum of cells incubated with benzpyrene or dibenzanthracene, the 423 nm (polycyclic hydrocarbon)/443 nm [NAD(P)H] ratio is 1.25 or over, while it decreases to 1.0 or less upon microelectrophoretic addition of glucose‐6‐phosphate [NAD(P) reduction].

Intracellular Accumulation of Rhodamine 110 in Single Living Cells

To gain a better understanding of the internalization of rhodamines, vital staining of living cells in situ by two different rhodamines, R110 and R123, was studied by microfluorometry. These dyes differ strongly in their lipophilic properties because of differences in charge distribution. Microspectrofluorometry was used to study the fluorescence emission spectra of R110-loaded cells to determine reliable loading conditions. Cell uptake and cell efflux studies of R110 were performed by numerical microfluorescence imaging. A slower uptake was observed for R110 (14 hr) vs R123 (2 hr), but the R110 efflux was much more rapid (30 min) than that of R123 (<24 hr). Although it appeared in the R110 and R123 co-localization study that R110 was able to accumulate in mitochondria, labeling with R110 was lower than with R123. Our results indicate that, rhodamine 110 in its acid cationic form is able to cross the plasma and mitochondrial membrane and to accumulate in cell compartments as does the cationic rhodamine 123. However, because of its acido-basic properties, R110 should be able to decrease the pH of cell compartments, depending on their ability to regulate pH. In such a model, mitochondrial pH should be more greatly decreased than cytosolic pH, leading to a lower mitochondrial accumulation of R110 than of R123. Surprisingly, these effects, which should affect the energetic state of mitochondria, do not influence cell growth, because no cytotoxic effect was observed. (J Histochem Cytochem 45:403–412, 1997)

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.