Studies of phenytoin binding to human serum albumin by high-performance affinity chromatography

Studies by biointeraction chromatography of binding by phenytoin metabolites to human serum albumin

“…The goal of this work is to study the binding of these metabolites to HSA by using high performance affi nity chromatography and columns containing immobilized HSA. Numerous reports using HPLC-based HSA columns have reported drug-binding properties that show good agreement with those seen for soluble HSA [7,14,15]; this includes the recent work performed with phenytoin in Reference [7].…”

“…As demonstrated previously [7,19], this situation is described by Equation (5) even if I (but not A) has multiple binding regions on the ligand. In this case, the value of K IL that is obtained from a plot of 1/k versus [I] gives the association equilibrium constant for I at its site of competition of A.…”

“…However, the reciprocal of the slope now gives the effective concentration of binding sites in the column (m L /V M ), and the ratio of the slope to the intercept provides K AL . This makes such an experiment valuable in determining both the association equilibrium constant for an analyte with L and the amount of ligand sites present for this analyte in an affi nity column [7,11,12,16].…”

“…When phenytoin is present in blood, it is highly bound to the carrier protein human serum albumin (HSA) [3,7]. A few reports have indicated that phenytoin interacts at the warfarinazapropazone site of HSA [8,9], while others have noted competition between phenytoin and digitoxin through the digitoxin site on HSA [10].…”

“…A few reports have indicated that phenytoin interacts at the warfarinazapropazone site of HSA [8,9], while others have noted competition between phenytoin and digitoxin through the digitoxin site on HSA [10]. Recently Chen et al have reported that phenytoin interacts at several sites on this protein [7], with association equilibrium constants at the indole-benzodiazepine and digitoxin sites of 1.04 (±0.05) × 10 4 and 6.5 (±0.6) × 10 3 M −1 at pH 7.4 and 37 °C; interactions involving allosteric effects and/or direct binding by phenytoin at the warfarin-azapropazone and tamoxifen sites of HSA were also noted.…”