Automated Flow Injection Gradient Technique for Binding Studies of Micromolecules to Proteins Using Potentiometric Sensors:  Application to Bovine Serum Albumin with Anilinonaphthalenesulfonate Probe and Drugs

An automated flow injection (FI) gradient technique is described for the binding study of the potentiometric probe 1-anilino-8-naphthalenesulfonate (ANS) to bovine serum albumin (BSA). Using a single-channel FI system with a mixing chamber and a flow ANS electrode, the binding parameters (binding constant and number of binding sites) were calculated using the Scatchard model. The concentration gradient was calibrated by injecting ANS in the stream, and the binding experiment was performed by injecting ANS−BSA solution in the carrier solution of equal albumin concentration. The equations describing the concentration gradient and the corresponding electrode potential curve are presented. A systematic study of the factors affecting the complexation equilibrium and the electrode response was performed. For the ANS binding to BSA, two binding classes were determined with binding constants of (2.1 ± 0.3) × 105 and (3.3 ± 0.8) × 103 M-1 and 3.8 ± 0.6 and 10 ± 2 binding sites per class, respectively, at 27 ± 1 °C, in 0.10 M phosphate pH 7.4. Competitive binding experiments of sulfamethoxazole, salicylate, azapropazone, ketoprofen, and tolmetin to albumin were also performed by monitoring ANS binding inhibition (decrease of apparent binding constant). This technique takes advantage of FI gradients and direct potentiometry and utilizes the total information contained in FI peaks, providing fast and accurate binding information in a wide range of concentration ratios.