PROTON, MG2-PROBE, MAG-INDO-1 - A FIRST STEP TO THE QUANTIFICATION OFINTRACELLULAR MG2+ CONCENTRATION( AND PROTEIN AS COMPETING LIGANDS FOR THE FLUORESCENT)

Increasing evidence of the role of magnesium in various cellular mecha nisms has led to the need to develop an accurate method for the evalua tion of magnesium concentration in cells. 1H-indole-6-carboxylic acid, 2-(4-bis[carboxymethyl]amino-3-[carboxy]ethoxy) (mag-indo-1) is used as a fluorescent indicator for ionized magnesium concentration. A phys icochemical study of this probe has pointed out (1) that at concentrat ions higher than 10 mu M, the presence of dimers can alter the differe nt equilibria and (2) at concentrations, avoiding the dimer (less than or equal to 10 mu M), three fluorescent forms are in equilibrium with the deprotonated form of mag-indo-1 (L), which are the protonated for m LH, the magnesium-bound form LM and the protein-bound form LP. A mod el is proposed that takes into account the equilibria between the four species. In a solution containing magnesium and protein, a complex fl uorescence spectrum can be resolved by a combination of the three fluo rescence spectra (L, LM, LP). However, under these conditions, the LH fluorescence spectrum is not taken into account for the spectral resol ution. Finally, from the contribution of characteristic fluorescence s pectra in the experimental fluorescence spectrum, the magnesium concen tration can be estimated with accuracy. Such a method should be furthe r applied to magnesium determination in different cell lines.