AFFINITY THERMOPRECIPITATION - CONTRIBUTION OF THE EFFICIENCY OF LIGAND PROTEIN-INTERACTION AND ACCESS OF THE LIGAND

Conjugates of two thermoprecipitating polymers, poly(N-vinyl caprolactam) and poly(N-isopropylacrylamide), with soybean trypsin inhibitor, Cibacron Blue 3GA, Cu-iminodiacetic acid, and p-aminobenzamidine were synthesized. The interaction of these conjugates with trypsin and lactate dehydrogenase was studied. Coupling of the ligand to a polymer resulted in a 100-1000-fold decrease in enzyme-affinity. Rough theoretical estimates revealed that a successful affinity precipitation required that the binding of a target protein and a ligand coupled to a polymer have binding constants on the order of 10(-7)-10(-8) M. Such strong affinity of low molecular weight ligands for proteins is quite unique. Affinity thermoprecipitation is restricted to those systems with ligands that can provide binding constants of 10(-9)-10(-11) M or alternatively multipoint attachment of the target protein molecule. The ligand in the ligand-polymer conjugate is still accessible to the protein after thermoprecipitation, and the latter can bind with the particles of the dispersion of thermoprecipitated ligand-polymer conjugate. The aggregation and formation of a bulk polymer precipitate may result in stripping of enzyme molecules from the surface of the particles.