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

本研究旨在开发一种使用50-100 μL样品准确测量蛋白质与低分子量化合物之间相互作用的方法。我们重新评估了正面凝胶色谱法（FGC），一种已建立的方法，直接测量蛋白质-配体混合物的游离配体浓度，可以使用填充有多孔凝胶的微柱和/或毛细管柱。修改普通的HPLC系统，以满足所需的实验条件的微型FGC（mFGC），我们已经成功地获得结合数据，使用少量的样品。mFGC开辟了测量蛋白质结合功能的途径。目的：探讨血清标志物hennas蛋白结合参数对临床诊断和个体化治疗的意义。治疗糖尿病等疾病，我们现在正在开发一种自动进行mFGC的系统。以下是清单装的主要结果。50-100 μL sacle mFG的建立采用内径0.5-1.0 mm，长7.5-10 cm的高效凝胶过滤柱。对泵的流量精度和压力要求很高。为了使用普通的UV-Vis HPLC监测器，在进入监测器之前将abate稀释10倍。所有的连通实验室和阀门的死腔都被最小化，以防止溶剂-简单边界的扩大。mFGC理论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的模拟.人血清白蛋白结合数据库的建立利用mFGC测定了人血清白蛋白与标记化合物的结合曲线，确定了whirl复合物的晶体结构.测定从100 μL大鼠血清中纯化的腐血清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.