Zonal high-performance affinity chromatography as a tool for protein interaction studies with special reference to the lipase-colipase complex.
The technique of zonal high-performance affinity chromatography applied to the lipase-colipase system (lipase B as eluted acceptor and colipase as silica-bonded ligand) gave qualitatively the same results as conventional affinity chromatography. The elution volume of the acceptor increases with decreasing load introduced at constant volume into the column of ligand-bonded silica. This led to the use of a mathematical treatment for calculating the dissociation constant (KD) of the lipase-colipase complex. The influence of some physical and chemical chromatographic parameters was studied. Increasing temperature and flow-rate reduced the affinity of lipase for colipase, whereas it was only slightly modified by increasing the ionic strength. The KD value was minimal and equal to 0.1 X 10(-6) M at pH 4.7 and 0.38 X 10(-6) M at pH 6.5, after correction for the flow-rate. The latter value is similar to that obtained by more conventional techniques. The absence of some marked KD modifications by ionic strength and the value of delta S for the complex association obtained by temperature studies suggest the intervention of mixed hydrophobic-ionic interactions in the formation of the lipase-colipase complex. Their respective importances are discussed.
D000818:Animals; D002846:Chromatography, Affinity; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008049:Lipase / Q000032:analysis*; D011506:Proteins / Q000032:analysis*; D012822:Silicon Dioxide; D013552:Swine; D013696:Temperature
D008024:Ligands; D011506:Proteins; D012822:Silicon Dioxide; D008049:Lipase