亲和毛细管电泳法测定牛血清白蛋白和加替沙星的结合常数

利用亲和毛细管电泳法对牛血清白蛋白(BSA)与加替沙星(GT)间的结合反应及其相互作用做了初步探索,并应用淌度比(M)作为指标测定了两者的结合常数。以20 mmol/L pH 7.4的磷酸盐缓冲液作为运行缓冲液,分别以GT和BSA作为添加剂,另一组分为进样样品,内标为二甲基甲酰胺,于214 nm波长下检测。两种测定条件下得到的结合常数分别为4.4×104 L/mol和4.2×104 L/mol,与传统的荧光淬灭法测得的结果基本一致。该方法具有简单、高效的优点。

Determination of the Binding Constant of Bovine Serum Albumin and Gatifloxacin Using Affinity Capillary Electrophoresis

The interaction between bovine serum albumin (BSA) and gatifloxacin (GT) was investigated using affinity capillary electrophoresis (ACE) method, mobility-shift method. The mobility ratio of the internal standard and the sample was used to calculate the binding constant K(b) of bovine serum albumin and gatifloxacin. At first, 20 mmol/L, pH 7.4 sodium phosphate buffer solution (PBS) added with 0 - 1 000 micromol/L GT as running buffer was used, BSA as the sample was used. K(b) calculated from this experiment was 4.4 x 10(4) L/mol. Then 0 - 12.5 micromol/L BSA was used as an additive to do the ACE experiment, the K(b) obtained was 4.2 x 10(4) L/mol. The two values matched well with each other. In order to improve the shape of the peak and restrain the adsorption of BSA to the internal wall of the quartz column, in the second method, 0.25 mol/L Gly and 0.5 mmol/L ethylenediamine tetracetate (EDTA) were added to the running buffer, and 0.5% sodium dodecyl sulfate (SDS) solution was used to rinse the column. Satisfactory effect was obtained. The K(b) from fluorescence method was also calculated with a value of 2.7 x 10(4) L/mol. This work demonstrated that the determination of the binding constant by ACE is simple with high performance.