Fluorometric determination of drug-protein association constants: binding of pamaquine by bovine serum albumin.

The binding of pamaquine to bovine serum albumin is accompanied by the enhancement of the fluorescence efficiency of the former but without shifting its fluorescence energy. This phenomenon was used to evaluate the stoichiometry and strength of the binding. The results indicate that three singly protonated pamaquine molecules are bound by each bovine serum albumine molecule. The individual binding constants were calculated by using the Bjerrum technique. The average values of the three constants were K1 = 6.4 X 10(7), K2 = 3.1 X 10(6), and K3 = 1.9 X 10(5), indicating that, compared to anionic drugs and fluorescent probes, pamaquine is very strongly bound by the protein.