Accurate measurement of the interaction of serum proteins with low-molecular weight compounds by micro-scale frontal gel chromatogrphy

本研究旨在建立一种在50-100 μL样品中精确测量蛋白质与低分子量化合物相互作用的方法。我们重新评估了正面凝胶色谱法（FGC），这是一种直接测量蛋白质-配体混合物中游离配体浓度的方法，可以使用填充有多孔凝胶的微柱和/或毛细管柱来去除FGC。通过对普通高效液相色谱系统进行改进，使其满足微尺度FGC （mFGC）所需的实验条件，我们成功地利用少量样品获得了结合数据。mFGC为测量scrum蛋白的结合功能开辟了道路。目的探讨血清蛋白的结合参数对诊断和个体化治疗是否有帮助。在糖尿病等疾病的治疗中，我们正在开发一种自动进行mFGC的系统。以下是主要结果的列表。建立50-100 μL粒径的mfghigh高效凝胶过滤柱，内径0.5 ~ 1.0 mm，长7.5 ~ 10 cm。对泵要求高精度的流量和严格的恒压。使用普通紫外-可见高效液相色谱监测仪时，稀释10倍后进入监测仪。所有连接实验室和阀门的死区都被最小化，以防止溶剂-简单边界的扩大。根据凝胶过滤柱的实际结构，建立了mFGC模型，并编制了计算机程序进行严格模拟。3 .建立人血清白蛋白结合数据库，利用mFGC测定了人血清白蛋白与标记化合物的结合曲线，测定了旋流复合物与白蛋白的晶体结构。测定100 μL大鼠血清中纯化的腐血白蛋白的旧结合曲线。

The present study aimed to develop a method to measure accurately the interaction between a protein and low-molecular weight compounds using 50-100 μL, of samples. We reevaluated frontal gel chromatography (FGC), an established method to directly measure the free ligand concentration of a protein-ligand mixture, to may out FGC using micro and/or capillary columns packed with porous gels. Modifying ordinary HPLC systems to meet the experimental conditions needed for the micro-scale FGC (mFGC), we have succeeded in obtaining binding data using small amounts of samples. mFGC opens the way to measure the binding function of scrum proteins. To examine whether the binding parameters of serum proteins for marker hennas are useful far diagnosis and personalized. Therapy of diseases such as diabetes mellitus we are now developing a system to carry out mFGC automatically. The following is a list attire main results.1. Establishment of 50-100 μL sacle mFGCHigh performance gel filtration columns with 0.5-1.0 mm internal diameter and 7.5-10 cm length were used. High accuracy of the flow rate and strictly constant pressure were required for pumps. To use ordinary UV-Vis HPLC monitor, the abate was 10-fold diluted before entering the monitor. All of the dead spaces of connecting labs, and valves were minimized to prevent the broadening of the solvent-simple boundaries.2. Theory of mFGCWe made a mFGC model based on the actual structure of the gel filtration column and developed a computer program for strict simulation.3. Building of the binding data base of human serum albuminUsing mFGC, we are now measuring the binding curves of human serum albumin for marker compounds, the crystal structures of whirl complex with albumin have been determined.4. Measurement oldie binding curves of rot serum albumin purified from 100 μL of individual rat serum.