Calibration of Phen Green for use as a Cu(I)-selective fluorescent indicator

Conditional stability constants for the Cu-sensitive fluorescent dye Phen Green™ SK (PGSK) were determined for complexes containing both Cu(I) and Cu(II). Experimental conditions were optimized to minimize oxidation of Cu(I) to Cu(II). A binding constant of 10^11.38 for PGSK and Cu(I) was determined using chloride as the competing ligand and an iterative procedure involving equilibrium calculations to fit experimental data. The new constant was tested by evaluating PGSK fluorescence in the presence of the strong Cu(I)-ligand neocuproine and thiourea. There was good agreement between experimental data and changes in fluorescence predicted by calculations using the new constant and published constants for the competing ligands. Using EDTA to buffer the free ion concentration of Cu²⁺, the conditional stability constant of PGSK with Cu(II) was measured to be 10^9.8. Both of these new constants are significantly higher than previously published values. Finally PGSK was used to examine the kinetics of Cu(I) dissociation from the biologically important ligand glutathione.