To minimize the concentration of chelate-conjugated macromolecules required to determine the number of metal binding sites, the terbiumdipicolinic acid complex (Tb(IlI):DPA) fluorescence quenching method of Ma, Hwang, and Lee (Pharmaceutical Research 10: 204-207, 1993) was used at a lower initial concentration of TWII1):DPA (0.2 |iM compared to 1.0 uM). Fluorescence quenching by either EOT A, diethylenetriaminepentaacetic acid (DTPA), poly (L-lysine)-DTPA or poly (L-lysine)-DTPA linked to mouse IgG (IgG-PI.-DTPA) was non-linear. Based on the law of mass action and non-linear curve fitting, the data suggests a three-site binding model for Tb(III):DPA complex formation in which the binding of the first and second DPA produce the majority of complex fluorescence. Initial binding of chelators to the complex results in only slight decreases in fluorescence with the majority of fluorescence quenching resulting from dissociation of more than one DPA from the complex. Although fluorescence quenching was non-linear, binding capacity of each chelator relative to either EDTA or DTPA (as suggested by Ma, et. al.) could be used to estimate the concentration of metal binding sites consistent with concentrations reported in the literature using radionuclides.
|State||Published - 1998|