Estimation of the binding affinity constants of soluble ligand-receptor complexes by a rapid filtration technique: [3H]-flunitrazepam-bovine serum albumin as an example.
A method for determining the equilibrium dissociation constant (KA) of a soluble ligand (L) from a soluble receptor (A) in the presence of another solid phase receptor (R) for the same ligand was developed. The total and nonspecific binding of L to R was measured in the presence and in the absence of A. The separation of bound and free L was done by a rapid filtration technique so that only the complex RL, but not AL, was recovered. An apparent dissociation constant (KR,app) was calculated from the saturation curve obtained in the presence of A. The magnitude of KA could be determined from this KR,app and the value of the equilibrium dissociation constant of the complex R-L (KR) calculated from the saturation curve in the absence of A. The equality of the Bmax values obtained in the presence and in the absence of A assured the accuracy in the determination of KA so that the fulfillment of this condition could be used as an internal control. For the correct definition of nonspecific binding, the displacement agent (L1) should be used at concentrations within the range 10(2).KR < L1 < 10. K4. This fact constraints the applicability of the method to systems where KA/KR > 10(3). The highest sensitivity of the method can be attained when 0.33 < [At]/KA < 3. The equilibrium binding constant of [3H]-flunitrazepam to non-delipidized bovine serum albumin determined by the present approach (31 +/- 7 mumol/L) did not differ significantly from the literature.
Perillo
M A
MA
Facultad de Ciencias Exactas, Físicas y Naturales, Universidad Nacional de Córdoba, Argentina.
Arce
A
A
D001665:Binding Sites; D001798:Blood Proteins / Q000378:metabolism; D003975:Diazepam / Q000378:metabolism; D005374:Filtration; D005445:Flunitrazepam / Q000378:metabolism*; D018757:GABA Modulators / Q000378:metabolism*; D008024:Ligands; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000378:metabolism*
D001798:Blood Proteins; D018757:GABA Modulators; D008024:Ligands; D011956:Receptors, Cell Surface; D012710:Serum Albumin, Bovine; D005445:Flunitrazepam; D003975:Diazepam