Multivalency of the partitioning species in quantitative affinity chromatography. Evaluation of the site-binding constant for the aldolase-phosphate interaction from studies with cellulose phosphate as the affinity matrix.
Theory is presented which describes the competitive interaction of a multivalent solute with a univalent ligand and an affinity matrix. The formulation accounts for cross-linking interactions of the multivalent solute, and of its complexes with ligand, with matrix interaction sites in terms of two site-binding constants pertaining respectively to ligand-solute and solute-matrix interactions. Explicit expression are derived which permit evaluation of these constants from experimental results obtained in partition equilibrium experiments or in frontal affinity chromatography studies. These relations are explored in partition equilibrium experiments conducted with cellulose phosphate as the matrix, aldolase as the solute, and phosphate as the ligand. At pH 7.4, I = 0.15, a value of 350 +/- 60 M-1 was obtained for the aldolase-phosphate site-binding constant, in close agreement with the corresponding value deduced from competitive inhibition studies. It is concluded that the present approach is particularly suited to the elucidation of weak interactions, which cannot be reliably studied by conventional means.
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D005634:Fructose-Bisphosphate Aldolase / Q000302:isolation & purification*; D008433:Mathematics; D009132:Muscles / Q000201:enzymology; D010710:Phosphates; D011485:Protein Binding; D011817:Rabbits
D010710:Phosphates; D005634:Fructose-Bisphosphate Aldolase