The binding of 8-anilino-1-naphthalenesulfonate to bovine serum albumin was reinvestigated. A fluorescence method was used to obtain the binding data which were treated appropriately to obtain the number of binding sites and the equilibrium constants. The results confirm that the number of binding sites for 8-anilino-1-napthalenesulfonate on bovine serum albumin is 3. The individual binding constants were calculated by using the Bjerrum technique. The average values of the three constants are K , = 2.9 X lo6, K2 = 6.1 X lo5, and K3 = 5.6 X lo5. 8-Anilino-1-naphthalenesulfonate (ANS) has been extensively used as a fluorescent probe for such studies as the quantitative analysis of serum proteins ( I ) , the study of the polarity of binding sites on proteins ( 2 ) , and the determination of the protein-binding parameters of other drugs by competitive binding (3, 4 ) . Rees and coworkers ( I ) reported the use of ANS for the assay of serum proteins.However, it was Daniel and Weber ( 5 ) who studied the interaction between ANS and bovine serum albumin (BSA) in detail, They reported that the interaction was reversible with an average binding constant of about lo6, and that five molecules of ANS were bound per molecule of BSA. Recently Ma, Jun, and Luzzi (6) have published new data on ANS-BSA binding. Their value for the average binding constant agrees well with that reported by Weber. However, they found that the maximum number of binding sites, N , on BSA, for ANS was three rather than five. Because of these conflicting results, it was decided to reinvestigate the ANS-BSA binding interaction.
EXPERIMENTALSodium 8-anilino-1-naphthalenesulfonate was purchased from Eastman Organic Chemicals, Rochester, N.Y. The sodium salt was purified by passing an aqueous solution of the salt through an Amberlite IR-120 (Mallinckrodt) column, and then further purified by crystallization from water. Crystalline bovine serum albumin of A grade (Pentex brand product of Miles Laboratory) was supplied by Calbiochem, San Diego, Calif. Commercially prepared BSA usually contains fatty acids as impurities and can be purified by acid charcoal treatment ( 7 ) . However, Chen ( 7 ) has shown that Pentex brand BSA contains only about 1 mole of fatty acid per mole of BSA. Moreover, Santos and Spector (8) have reported that up to 2 moles of long chain fatty acid per mole of BSA do not affect the ANS-BSA binding. Consequently, the pretreatment of BSA with acid charcoal was deemed unnecessary. Because of the hygroscopic nature of BSA, it has been common practice not to accurately weigh samples routinely for dissolution in water, but rather to photometrically determine the absorbance at 280 nm of aqueous solutions containing roughly weighed BSA samples. The concentration in g/l. is obtained from Beer's law, assuming a value of E ;Trn of 6.60 ( 4 ) or in moles/l. by assuming as well the average molecular weight of 69,000 (9, IO) so that c = 4.55 X IO4 at 280 nm. In our experience, during the course of an average accurate weighing (in the extremely humid ...