A displacement approach for competitive drug-protein binding studies using the potentiometric 1-anilino-8-naphthalene-sulfonate probe technique.

A displacement approach for competitive binding studies was developed. The method utilizes the potentiometric 1-anilino-8-naphthalene-sulfonate (ANS) probe technique and is applied to the binding study of several non-steroidal anti-inflammatory drugs (NSADs) to bovine serum albumin (BSA). A home-made ANS electrode was used to monitor the displaced free ANS probe from its binding sites on the protein molecule by the stepwise addition of the studied drug. To assess and compare quantitatively the displacing ability of the various drugs, the 'ANS Displacement Index' is used. The possible interference of 19 ionizable drugs (NSADs, sulfonamides, etc.) to the ANS selective electrode at pH 7.4 was studied and their potentiometric selectivity coefficients (K(pot)(ANS,D)) were determined. Correction procedures for the determination of the free ANS concentration are proposed in the case of interfering ionic drugs. A blank binding experiment in conjunction with the incorporation of K(pot)(ANS,D) values in the 'general competitive site oriented model' allows one to derive estimates for the drug binding parameters, i.e. the number of binding sites and association constants.

General treatment of competitive binding as applied to the potentiometric ion probe technique: application to the interaction of nonsteroidal anti-inflammatory drugs with bovine serum albumin.

The binding of naproxen, ketoprofen, phenylbutazone, salicylic acid, azapropazone, and indobufen to bovine serum albumin was studied by applying the potentiometric ion probe technique. An ion-selective electrode for the ion probe 1-anilino-8-naphthalene-sulfonate was utilized for the purposes of this study. A modified site-oriented competitive binding model was used for the estimation of the drugs' binding parameters, considering different number of binding sites on the competing binding class(es) for the probe and the drug. Calculations were based exclusively on the concentration data of the free probe. The model's ability for accurate estimations of binding parameters was evaluated by simulation studies. The following values of binding parameters were found at 25 degrees C for the drugs under study; naproxen, n1 = 9.1, k1 = 9.4 x 10(5) M-1; ketoprofen, n1 = 8.8, k1 = 10.8 x 10(5) M-1; phenylbutazone, n1 = 3.2, k1 = 1.4 x 10(5) M-1; salicylic acid, n1 = 2.6, k1 = 1.8 x 10(5) M-1, n2 = 21.5, k2 = 1.0 x 10(4) M-1; azapropazone, n1 = 0.5, k1 = 7.8 x 10(5) M-1, n2 = 26.3, k2 = 1.9 x 10(4) M-1; indobufen, n1 = 5.8, k1 = 5.8 x 10(5) M-1, n2 = 19.9, k2 = 3.8 x 10(5) M-1, where ni the number of binding sites of the i class and ki the corresponding association constant.

Use of 1-anilino-8-napthalenesulphonate as an ion probe for the potentiometric study of the binding of sulphonamides to bovine serum albumin and plasma.

The binding of sulphafurazole, sulphamethizole and sulphamethoxazole to bovine serum albumin (BSA) and human plasma has been studied in-vitro by potentiometry using an electrode selective for the ion probe 1-anilino-8-napthalenesulphonate (ANS). The method requires two separate potentiometric titrations of the binder solution with ANS in the absence and in the presence of sulphonamides. ANS displaced sulphonamides from the first-class of binding sites of both binders. The binding constants for sulphonamide-BSA interactions were higher than those for sulphonamide-human plasma. The expansion of the least linear limit of the response curve of the electrode down to 10(-7) M in the presence of BSA was also demonstrated. The reverse reaction, i.e. the displacement of the probe from the binding sites induced by the sulphonamides, was also explored. The proposed method is suitable for studying competitive binding interactions in biological specimens.