SEMI-MICRO-SCALE FRONTAL GEL CHROMATOGRAPHY OF INTERACTING SYSTEMS OFA PROTEIN AND SMALL MOLECULES - BINDING OF WARFARIN, TRYPTOPHAN, OR FMN TO ALBUMIN, AND OF O-NITROPHENOL TO CATECHOL 2,3-DIOXYGENASE

Frontal gel chromatography is a convenient and accurate method to obta in the free ligand concentration of a protein-ligand mixture, Because a large amount of sample (more than 6 ml) is required for the method, it has been rarely used for binding experiments, We have developed a s ystem to carry out frontal gel chromatography on a semi-micro scale us ing short gel filtration columns (4.6 mm X 50-100 mm); frontal chromat ograms could be obtained with small amounts of samples (1-2.5 ml) with in 20 min. We used this technique to examine the binding of warfarin, L-tryptophan, or FMN to human serum albumin, the binding of warfarin t o bovine serum albumin, and the interaction of catechol 2,3-dioxygenas e with o-nitrophenol. The data fitted to a binding model in which a pr otein has one or several independent binding sites, Both human and bov ine serum albumin showed the high-affinity bindings of two warfarin mo lecules. The binding number for L-tryptophan on human serum albumin wa s confirmed to be one, whereas maximal binding of FMN was 0.6 molecule per albumin molecule, o-Nitrophenol showed high-affinity binding only to holocatechol 2,3-dioxygenase, The absorption spectrum of the bound o-nitrophenol resembled that of anionic o-nitrophenol, These results demonstrated that frontal gel chromatography on a semi-micro scale is useful for the study of binding systems; the method is rapid and would be easy to automate fully.