Binding Study of Phenformin with Bovine Serum Album Using a Combined Technique of Equilibrium Dialysis with Flow-Injection Chemiluminescence Detection

ABSTRACT

The interaction between phenformin hydrochloride and bovine serum albumin (BSA) was investigated by the methods of chemiluminescence combined with equilibrium dialysis technique. A novel N-bromosuccinimide (NBS)–eosin Y (EY) chemiluminescence (CL) method was established for the determination of phenformin. The mechanism of this chemiluminescence system was proposed. Optimization studies were performed to determine the phenformin. Under the optimal conditions, the CL intensity was linear for a phenformin concentration over the range of 4.6 × 10⁻⁸ to 5.0 × 10⁻⁵ g/mL. The detection limit was 1.5 × 10⁻⁸ g/mL. The data obtained by the present equilibrium dialysis–CL system were analyzed using the Klotz plot and the Scatchard analysis. The results showed that the Klotz plot and the Scatchard plot are linear with good correlation coefficient, indicating that the phenformin has only one type of binding site on BSA. The binding parameters were the number of the binding sites n (1.02) and the estimated association constant K (2.66 × 10⁴ L/mol). The chemiluminescence system combined with equilibrium dialysis developed in this work demonstrated its use for determination of interaction between drug and protein by using relatively simple instrument.

KEYWORDS:

• chemiluminescence
• drug–protein interaction
• eosin Y
• equilibrium dialysis
• N-bromosuccinimide
• phenformin hydrochloride

ACKNOWLEDGMENTS

This work was supported by the National Natural Science Foundation of China (no. 20973149), the Department of Science and Technology of Shandong Province (no. 2009GG10009053), and the Outstanding Youth Natural Science Foundation of Shandong Province (no. JQ201006).