Protein binding to two-dimensional hydrophobic binding-site lattices: Sorption kinetics of phosphorylase b on immobilized butyl residues

Abstract

The adsorption and desorption kinetics of the binding of the large ( m = 200 kDa) protein, phosphorylase b, to a hydrophobic surface (butyl agarose) was studied. The initial adsorption rate is a hyperbofic function of the initial free protein concentration (saturation kinetics). At 5°C the apparent dissociation constant for the half-maximal rate ( K d ) is 13.2 μ M. A maximal rate ( V max) of 43 μmole/(liter packed agarose × min) is obtained. The first-order rate constant of the rate-limiting step of adsorption ( k 2) is 1.8 × 10 −3 s −1 ( t 1 2 ∼ 6 min) and illustrates that the adsorption of this protein to a hydrophobic surface is a slow process. It is concluded that adsorption is a multistep reaction and that the rate-limiting step is not the collision rate but a slow conformational change or reorientation step of the protein on the butyl agarose surface. From the initial desorption rates, measured in dilution experiments, the apparent, first-order off-rate constants k′ off) were determined. It is shown that the basal, off-rate constant ( k′ off ∼ 64 × 10 −6 s −1; t 1 2 ∼ 180 min) obtained with butyl agarose having a surface concentration ca. 320 nmole butyl residues/m 2, does not show the constancy expected for the binding of a protein to single, independent sites on a surface. In fact, the off-rate constant depends on the fractional saturation of the adsorbent surface with protein and is sensitive to the presence of other protein(s) in the bulk. It is concluded that here is one of the first examples of a protein showing negative cooperative surface kinetics on an adsorbent.