Improved Frontal Gel Filtration Chromatography to Examine the Interaction between Small Molecules and a Protein with Multiple Specific Binding Sites

Interaction between small molecules (ligands) and a protein is the basis of protein function and drug action. Therefore, it is essential to know the number and binding constants of specific binding sites that are most relevant to protein functions. These binding parameters can be estimated by the analysis of the average number of bound ligands per protein (<i>r</i>) as a function of free ligand concentration ([L]_f). Frontal gel filtration chromatography (FGC) is a well-established method to obtain a fraction of free ligand from a protein-ligand mixture without any disturbance of the original binding equilibrium of the mixture. The ligand concentration of this fraction gives [L]_f, and <i>r</i> is obtained as the ratio of the difference between the total ligand concentration and [L]_f to the total protein concentration. The serious drawback of conventional FGC is large sample consumption to perform a single experiment. Here, we improved FGC using microbore columns packed with gel matrix (particle size 4 μm, pore size 12.5 nm) and an automated injection system to reduce sample consumption to 100 μL levels. Its performance was tested on the interaction of 5 vertebrate serum albumins with aromatic compounds of methyl orange, dabsyl amino acids, and <i>S</i>- and <i>R</i>-warfarin. A subtle chiral difference in the warfarin binding was clearly detected for human serum albumin. A large species difference in <i>S</i>-warfarin binding to albumin was also found. Further scale-down of FGC with a capillary column is suggested to be possible by simulation using the program developed in the present study, promising broader application of FGC to various binding systems.