Analysis of nonlinear quenching of terbium(III): dipicolinic acid complex fluorescence by chelators and chelate-conjugated macromolecules

The terbium-dipicolinic acid complex (Tb(III):DPA) fluorescence quenching m ethod of Ma, Hwang, and Lee [Pharm. Res. (1993) 10, 204-207] was used at a lower initial concentration. of Tb(III):DPA (0.2 mu M compared to 1.0 mu M) to minimize the concentration of chelate-conjugated macromolecules require d for estimating concentrations of metal-binding sites. Fluorescence quench ing I,by either EDTA, diethylenetriaminepentaacetic acid (DTPA), poly (L-ly sine)-DTPA, or poly (L-lysine)-DTPA linked to mouse IgG (IgG-PL-DTPA) was n onlinear, suggesting initial binding of chelator results in minimal change in Tb(III):DPA complex fluorescence. On the basis of the law of mass action and nonlinear curve fitting, the data suggests a three-site binding model for Tb(III):DPA complex formation in which the binding of the first and sec ond DPA produce the majority of complex fluorescence. Initial binding of ch elators to the complex results in only slight decreases in fluorescence wit h the majority of fluorescence quenching resulting from dissociation of mor e than one DPA from the complex. Although fluorescence quenching was nonlin ear, 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 meta l-binding sites consistent with concentrations reported in the literature u sing either radionuclides or nuclear magnetic resonance. Estimation of chel ating groups on chelate-conjugated macromolecules at lower concentrations w ould minimize the amount of sample required for analysis during synthesis a nd isolation of chelate-conjugated macromolecules. The suggested model for the stoichiometry of Tb(III):DPA complex fluorescence may be useful in furt her refinement of methods for estimating chelator concentrations and for de sign of fluorescent probes for chelate-conjugated macromolecules.