Semi-micro-scale frontal gel chromatography of interacting systems of a 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 obtain 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 system to carry out frontal gel chromatography on a semi-micro scale using short gel filtration columns (4.6 mm x 50-100 mm); frontal chromatograms could be obtained with small amounts of samples (1-2.5 ml) within 20 min. We used this technique to examine the binding of warfarin, L-tryptophan, or FMN to human serum albumin, the binding of warfarin to bovine serum albumin, and the interaction of catechol 2,3-dioxygenase with o-nitrophenol. The data fitted to a binding model in which a protein has one or several independent binding sites. Both human and bovine serum albumin showed the high-affinity bindings of two warfarin molecules. The binding number for L-tryptophan on human serum albumin was 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.
Honjo
M
M
Department of Orthopaedic Surgery, Shiga University of Medical Science, Ohtsu.
Ishida
T
T
Horiike
K
K
D000818:Animals; D050557:Catechol 2,3-Dioxygenase; D002417:Cattle; D002850:Chromatography, Gel / Q000379:methods*; D049308:Dioxygenases; D005486:Flavin Mononucleotide / Q000378:metabolism; D006801:Humans; D008024:Ligands; D009596:Nitrophenols / Q000378:metabolism; D010105:Oxygenases / Q000378:metabolism*; D011485:Protein Binding; D012709:Serum Albumin / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000378:metabolism; D014364:Tryptophan / Q000378:metabolism; D014859:Warfarin / Q000378:metabolism*
D008024:Ligands; D009596:Nitrophenols; D012709:Serum Albumin; D012710:Serum Albumin, Bovine; D014859:Warfarin; D005486:Flavin Mononucleotide; D014364:Tryptophan; D010105:Oxygenases; D049308:Dioxygenases; D050557:Catechol 2,3-Dioxygenase