Heterogeneous binding of aldolase to phosphocellulose: Interpretation in terms of a “concerted cluster” model of multivalent affinity

Abstract

It has been suggested that when multi-site protein molecules bind to immobilised ligands on an inflexible matrix, they should encounter discrete sets of single and clustered ligands, binding to clusters being concerted and very tight [R.J. Yon, J. Chromatogr., 457 (1988) 13–23]. To test this model, the aldolase-phosphocellulose interaction was re-examined at low protein concentrations, with and without the presence of soluble ligands. In all cases the data plotted as non-linear (concave upwards) Scatchard plots, indicating at least two populations of adsorption sites, while soluble ligands produced competitive effects as expected. When fitted to a 2-population Langmuir model based on the concerted concept, the data suggested that (a) a very small proportion (about 0.3%) of the total immobilised phosphate was accessible as matrix ligand; (b) of this, about 8% comprises accessible pairs; (c) the matrix ligand was non-randomly distributed within the actual matrix volume; (d) affinity constans for soluble ligands were close to their published values, and (e) the effective matrix ligand is a biphosphate structure in phosphocellulose. It is suggested that the concerted-cluster model may be valid for an affinity system based on a “hard” matrix such as cellulose.