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

点击翻译按钮获取中文摘要

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.