Studies on the interaction of a surface-bound ligand with a multi-valent high-molecular-weight ligate : The biotinycellulose—avidin system

Abstract

Two types of biotinylated cellulose disks were examined: filter-paper disks to which biotin had been covalently attached directly to the paper surface (biotinylcellulose) and disks on which biotin was attached to polyacrylamide side-chains grafted onto the filter-paper surface (biotinylpolyacrylamide—cellulose). The amount of avidin taken up from solution by these disks was linearly related to the avidin input concentration and could be estimated by exposure to [ 14C]biotin. The avidin-binding capacity of the disks depended on the surface density of covalently attached ligand and exhibited hyperbolic, Langmuir-type behaviour for both types of disks. The [ 14C]biotin binding capacity of avidinylated disks, on the other hand, showed anomalous, biphasic behaviour: at higher ligand densities, a decrease in [ 14C]biotin binding was observed. The largest anomalies were obtained with biotinylpolyacrylamide—cellulose disks. Calculated ratios of bound [ 14C]biotin vs. amount of avidin tetramer (B/A 4) showed a similar biphasic behaviour. A constant value of B/A 4 = 3 was obtained at low ligand densities, whereas B/A 4 decreased monotonously with increasing ligand density and asymptotically approached B/A 4 = 1. The data could be explained by assuming that at high ligand densities tetrameric avidin interacts with more than one surface-bound biotin residue.