Protein binding to two-dimensional hydrophobic binding-site lattices: adsorption hysteresis on immobilized butyl-residues

Long-lived metastable states involving multiple binding sites of a protein ligand with immobilized alkyl residues on a solid phase can be observed at high ionic strength between butyl agaroses (5.21 μ mol/ml packed gel) and phosphorylase b by perturbations enforcing either the on-reaction (adsorption) or the off-reaction (desorption). These apparent equilibrium states are suggested because the adsorption isotherms of phosphorylase b on butyl agaroses are not retraced by the desorption isotherms. In this first example of macromolecular adsorption hysteresis on immobilized alkyl residues, it can be shown that the irreversible entropy (Δ_iS) produced in an adsorption-desorption cycle lies between 6 (5 μ mol/ml packed gel) and 40 (21 μ mol/ml packed gel) J mol ¹ K⁻¹. For the latter gel the apparent standard entropy of adsorption (Δ_aS_i⁰′) is 160 J mol⁻¹ K⁻¹. The metastable state observed during adsorption is probably due to an energy barrier which must be overcome for the nucleation of protein binding on the matrix. Other metastable states may possibly be encountered during desorption when the adsorbed enzyme resists the breakage of hydrophobic interactions. In the transition from the adsorption branch to the desorption branch of the hysteresis loop, the apparent affinity of the enzyme-matrix interaction is enhanced. For the desorption branch, the apparent association constant of half-maximal saturation corresponds to K_d,0.5′ = 4.2 × 10⁹ ]m⁻¹ as compared to the respective constant of adsorption K_{a, 0.5}′ = 1.6 × 10⁵m⁻¹ (gel: 21μ mol/ml packed gel). Since the area of the hysteresis loops (see also Δ_iS) depends strongly on the density of butyl residues on the gel, it is concluded that the number of alkyl residues interacting with the protein molecule is crucial for the metastable states and hysteresis. It is unlikely that hysteresis is due to the pore structure of the agarose or to nearest neighbour interactions of ligand molecules. Since thermodynamic irreversibility and hysteresis may be encountered when macromolecules, such as proteins, are adsorbed to cell membranes or cell organelles: an analysis and understanding of these phenomena should be of general biological significance.