title: Development of a fluorescence model for the determination of constants associated with binding, quenching, and Förster resonance energy transfer efficiency.
abstract: Determining accurate dissociation constants for equilibrium processes involving a fluorescent mechanism can prove to be quite challenging. Typically, titration curves and nonlinear least squares fitting of the data using computer programs are employed to obtain such constants. However, these approaches only consider the total fluorescence signal and often ignore other energy transfer processes within the system. The current model considers the impact on fluorescence from equilibrium binding (viz., metal-ligand, ligand-substrate, etc.), quenching, and resonance energy transfer. This model should provide more accurate binding constant as well as insights into other photonic processes. The equations developed for this model are discussed and are applied to experimental data from titrimetric experiments. Since the experimental data are generally in excess of the number of parameters that are needed to define the system, fitting is operated in an overdetermined mode and employs error minimization (either absolute or relative) to define goodness of fit. Examples of how changes in certain parameters affect the shape of the titrimetric curve are also presented. The current model does not consider chelation-enhanced fluorescence.
authors: Department of Chemistry and Biochemistry, The University of Texas at Austin, Austin, TX 78712, United States; Sapling Learning, Austin, TX, United States.
authors: Shelly L
authors: SL
authors: Casciato
authors: Department of Civil, Architectural and Environmental Engineering, The University of Texas at Austin, Austin, TX 78712, United States.
authors: Howard M
authors: HM
authors: Liljestrand
authors: Department of Chemistry and Biochemistry, The University of Texas at Austin, Austin, TX 78712, United States. Electronic address: holcombe@mail.utexas.edu.
authors: James A
authors: JA
authors: Holcombe
keywords: Analysis; FRET; fluorescence; model; quenching; simulation