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.
Hirshfield
K M
KM
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
Toptygin
D
D
Packard
B S
BS
Brand
L
L
D000634:Aminoquinolines / Q000032:analysis* / Q000737:chemistry; D002118:Calcium / Q000032:analysis* / Q000737:chemistry; D005456:Fluorescent Dyes / Q000032:analysis* / Q000737:chemistry; D007700:Kinetics; D009930:Organic Chemicals; D013050:Spectrometry, Fluorescence / Q000379:methods
D000634:Aminoquinolines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C071336:calcium green; C035739:Quin2; D002118:Calcium