生物功能化色谱技术研究胰岛素与其受体间的相互作用

Analysis of interaction between insulin and its receptor by biofunctional chromatography

摘要:

生物功能化色谱技术是通过模拟细胞膜表面环境，在色谱系统内实现研究生物分子间相互作用过程的新技术。利用生物功能化色谱方法研究了胰岛素与其受体间的相互作用。将提取出的胰岛素受体混合物固定化在人工膜(Immobilized artificial membrane, IAM)的表面，并确定了固定化的最佳pH值为7.2，最佳离子强度为20 mmol，最佳有机溶剂浓度为2% (v/v)。通过区带洗脱法确定了胰岛素受体的存在及其与胰岛素间存在相互作用；通过前沿分析法确定了胰岛素受体与胰岛素间相互作用的结合常数大小约为0.701 nmol/L。

Abstract:

We used biofunctional chromatography to study the interaction between insulin and immobilized insulin receptor(IR). The heterogeneous insulin receptor extract from Wistar mouse liver was immobilized onto the surface of an immobilized artificial membrane through coating. The detergent was removed by dialysis to reduce its interference with the chromatography. To optimize the protein immobilization, we optimized the coating conditions at: pH 7.2, ion strength 20 mmol, and organic solvent 2% (v/v). The competition and self-displacement interacting study of insulin with IR was conducted by the zonal elution using insulin as the self-competition agent, the result indicated that the insulin receptor was active and interacted with insulin. In addition, relative binding constant was determined by frontal analysis by changing the concentration of insulin in the mobile phase.