title: Spectroscopic analysis of ligand binding to lanthanide-macrocycle platforms.
abstract: A high-affinity, binary Eu(3+) receptor site consisting of 1,4,7,10-tetraazacyclododecane-1,7-diacetate (DO2A) was constructed with the goal of improving the detection of dipicolinic acid (DPA), a major component of bacterial spores. Ternary Eu(DO2A)(DPA)(-) complex solutions (1.0 microM crystallographically characterized TBA x Eu(DO2A)(DPA)) were titrated with EuCl3 (1.0 nM-1.0 mM); increased Eu(3+) concentration resulted in a shift in equilibrium population from Eu(DO2A)(DPA)(-) to Eu(DO2A)(+) and Eu(DPA)(+), which was monitored via the ligand field sensitive (5)D0 --> (7)F3 transition (lambda(em) = 670-700 nm) using luminescence spectroscopy. A best fit of luminescence intensity titration data to a two-state thermodynamic model yielded the competition equilibrium constant (Kc), which in conjunction with independent measurement of the Eu(DPA)(+) formation constant (Ka) allowed calculation of the ternary complex formation constant (Ka'). With this binding affinity by competition (BAC) assay, we determined that Ka' = 10(8.21) M(-1), which is approximately 1 order of magnitude greater than the formation of Eu(DPA)(+). In general, the BAC assay can be employed to determine ligand binding constants of systems where the lanthanide platform (usually a binary complex) is stable and the ligand bound versus unbound states can be spectroscopically distinguished.
authors.affiliation: Planetary Science Section, Jet Propulsion Laboratory, Pasadena, California 91109, USA. james.p.kirby@jpl.nasa.gov
authors.forename: James P
authors.initials: JP
authors.lastname: Kirby
authors.forename: Morgan L
authors.initials: ML
authors.lastname: Cable
authors.forename: Dana J
authors.initials: DJ
authors.lastname: Levine
authors.forename: Harry B
authors.initials: HB
authors.lastname: Gray
authors.forename: Adrian
authors.initials: A
authors.lastname: Ponce
mesh_terms: D001665:Binding Sites; D028581:Lanthanoid Series Elements / Q000737:chemistry*; D008024:Ligands; D008163:Luminescent Measurements; D009942:Organometallic Compounds; D010848:Picolinic Acids / Q000032:analysis* / Q000737:chemistry; D013057:Spectrum Analysis; D013171:Spores, Bacterial / Q000737:chemistry; D013816:Thermodynamics
chemical_list: D028581:Lanthanoid Series Elements; D008024:Ligands; D009942:Organometallic Compounds; D010848:Picolinic Acids; C004860:dipicolinic acid