Dynamic fluorescence measurements of two-state systems: applications to calcium-chelating probes

A fluorescence technique for characterizing ligand binding is evaluated. This technique uses the combination of steady-state and time-resolved methods to recover molar concentrations and overcomes errors inherent in the use of either method alone. The technique is applicable to time-resolved measurements made either with time-domain or frequency-domain instrumentation. A straightforward single-frequency phase/modulation approach is presented to determine whether an experimental system can be described by a two-state system. The approach is based on a nonlinear transformation of the phase/modulation data that results in a linear model function. Here, the theory is applied to the fluorescent calcium-binding probes Quin-2 and Calcium Green, but is relevant to studies involving other interacting systems. The technique described is used to assess the fraction of bound ligand (Ca2+) and binding constants for these probes.