Protein interaction with immobilized ligands: quantitative analyses of equilibrium partition data and comparison with analytical chromatographic approaches using immobilized metal affinity adsorbents.

摘要: Quantitative or analytical affinity chromatography has been successful primarily for the analysis of biologically determined macromolecular relationships. approaches are also needed to better characterize simpler, chemically defined immobilized ligands with potential selective interaction specific, predetermined protein surface groups. Protein metal is a rather and versatile, high-affinity adsorption technique which there little quantitative information. Using model interactions Cu2+ ions, we have compared frontal chromatographic methods simple, nonchromatographic protocol rapid determination Values obtained equilibrium dissociation constant (Kd) binding capacity (Lt) characterizing lysozyme were quite similar by (Kd = 37-42 X 10(-6) M; Lt 6.8-7.4 mol protein/ml gel) analyses 33 +/- 4.7 5.8-6.1 gel; 14 determinations). The ovalbumin was characterized an 4.2-4.8 M) (Lt 1.5-2.1 same regardless method analysis. These values indicate that total bound at saturation corresponds as much 17% ions (approximately 40 mol/ml gel). Thus, depending on fraction available given (e.g., lysozyme), number individual actively participating well those rendered unavailable upon events may be greater than 1. Linear Scatchard plots both (purified) suggest presence only single type Cu2+-protein operative under experimental conditions employed. However, data demonstrated ability simultaneously resolve contribution two components whose predicted chromatography. Our results support validity utility analyzed according equations outlined others alternative methods.