For the study of biological systems such as living cells, access to oxygen concentrations in various organelles is important. In living cells, the lifetime of pyrene fluorescence can be used to measure local oxygen concentrations. We designed and synthesized a new probe to measure the oxygen concentration in mitochondria ([1'- pyrene butyl]-2-rhodamine ester, PRE). The localization of the probe was studied by videomicrofluometry in 3T3 cells and confirmed to label the mitochondria. We compared the lifetime of PRE with the well-known cytosol specific probe Pyrene Butyric Acid in (1) in living cells (2) in liposomes and (3) in solution. Liposomes were used to investigate the effect of phospholipid bilayer organization on the fluorescence lifetimes. Depending on the oxygen concentration we observed lifetime variation ranges of (1) 60 ns (hyperoxygenation) - 110 ns (anoxy) in cells (2) 60 - 220 ns in liposomes and (3) 6 - 220 ns in ethanolic solution. These results indicate that, under hyperoxygenation, quenching is less efficient in organized environments than in solution. Without oxygen and in cellular medium, the quenching depends on the composition of the probe environment. Accordingly, these probes can be used to measure the intracellular oxygen concentrations as well as changes in the environment.
Magnesium complexation with Mag-indo-1 results in a shift of the emission fluorescence spectrum from 480 nm to 417 nm. Mag-indo-1 is also able to bind calcium and zinc. These cationic interactions induce the same spectral shift but the fluorescence intensity and the dissociation constant are dependent of the nature of the cation. Furthermore Mag-indo- 1 bind also proteins through a specific interaction with some histidin residues. That interaction induces a characteristic spectral shift of the emission fluorescence spectra from 480 to 457 nM.
Mag-indo-1 is a well known fluorescent probe. Magnesium complexation results in a shift of the emission fluorescence spectrum from 480 nm to 417 nm with an intensity proportional to the magnesium concentration in the range 0.6 to 30 mM. Although designed as a specific magnesium chelator, Mag-indo-1 is also able to bind calcium and zinc. All these cationic interactions induced the same spectral shift but the fluorescence intensity and the dissociation constant are dependent of the nature of the cation. Furthermore Mag-indo-1 can also bind proteins through a specific interaction with some histidin residues. That interaction induces a characteristic spectral shift of the emission fluorescence spectra from 480 to 457 nM. All these properties suggest that Mag-indo-1 could be used to study the protein-cation binding. Emission and synchronous fluorescence techniques have been used to monitor that interaction with proteins such as bovine serum albumin, human serum albumin, turkey white egg lysozyme. Using a method of resolution of complex fluorescence spectra, it has been possible to calculate the number of interaction sites and the correlative dissociation constants. Depending on the nature of the protein a quenching of the natural fluorescence of the protein was observed, associated with an energy transfer from some tryptophan(s) to Mag-indo-1. All these data were tentatively correlated with the available information on the 3D conformation of the proteins. These results suggest that Mag-indo-1 could be used as an intramolecular fluorescent ruler to monitor the changes in 3D conformation of specific sub-domains of proteins.
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