Flow Injection on Line Oxidizing Fluorometry Coupled to Microdialysis Sampling for Studying Phentolamine-Bovine Serum Albumin Interaction

Abstract

Determination of phentolamine-bovine serum albumin (BSA) interaction based on a flow-injection microdialysis sampling fluorescence system (FI-MD-FL) was proposed. The analytical procedure was based on the oxidation of phentolamine by acidic Ce(IV) and monitoring of the fluorescence intensity of the formed Ce(III). The drug of phentolamine and BSA were mixed in different molar ratios in Ringer’s solution and incubated in a water bath. The microdialysate samples were on-line merged with acidic Ce(IV) solution by putting a microdialysis probe into the mixed solution and perfused with Ringer’s solution at 10 μL min-1. Then the sample was reached the flow cell, excited and monitored at 256/355nm (λex/λem). The dialytic efficiency under the experimental conditions was 38.8±2.2% (n=3). The data obtained was analyzed with Scatchard analysis and Klotz plot. The estimated association constant (K) and the number of the binding site (n) on one molecule of BSA by Scatchard analysis were 1.78×105 L mol-1 and 0.69, respectively. The proposed method has been applied to the study of the binding of phentolamine to bovine serum albumin (BSA) in vitro. The method provided a fast and simple technique for the study of drug-protein interactions.