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 str ucture bind proteins. It was suggested that these fluorescent probes are va luable tools to gain information on protein structure through the energy tr ansfer from tryptophans to the bound probe. Here, the binding of the fluore scent 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 (K -d = 2.8 x 10(-4) M for zero ionic strength) which can also interact with c alcium cations (K-d = 7.5 x 10(-7) M). Probe complexation with protein resu lts in a shift of the emission fluorescence spectrum of the probe from 480 to 457 nm. We used emission fluorescence techniques to monitor this interac tion. Computational resolution of the complex fluorescence spectra and a ne w software to test the theoretical model were developed in our laboratory. This enabled us to calculate the number of interacting sites and the dissoc iation constants. The fluorescent probe Mag-Indo-1 binds at a singular site with high affinity (K-d = 1.8 x 10(-7) M) to bovine serum albumin (BSA). S ince proteins are known to bind several compounds unspecifically, we have s tudied the influence of EDTA as a competitor of the probe. Our findings sug gest that the BSA binding site is identical for both Mag-Indo-1 and EDTA. W e found that EDTA binds the protein with K-d = 0.4 x 10(-3) M. We studied t he influence of calcium and found that Mag-Indo-1 does not bind the calcium free Apo-protein anymore. (C) 1999 Elsevier Science B.V. All rights reserv ed.