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.
Ribou
A C
AC
Physical Chemistry Laboratory, University of Perpignan, France. ribou@univ-perp.fr
Vigo
J
J
Viallet
P
P
Salmon
J M
JM
D000465:Algorithms; D001059:Apoproteins / Q000737:chemistry; D002118:Calcium / Q000737:chemistry*; D002614:Chelating Agents / Q000737:chemistry*; D004492:Edetic Acid / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D007211:Indoles / Q000737:chemistry*; D008954:Models, Biological; D011485:Protein Binding; D012710:Serum Albumin, Bovine / Q000737:chemistry*
D001059:Apoproteins; D002614:Chelating Agents; D005456:Fluorescent Dyes; D007211:Indoles; C088692:2-(4-(biscarboxymethyl)amino-3-(carboxymethoxy)phenyl)-1H-indole-6-carboxylic acid; D012710:Serum Albumin, Bovine; D004492:Edetic Acid; D002118:Calcium