Affinity thermoprecipitatin: Contribution of the efficiency of ligand–protein interaction and access of the ligand

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Abstract
Conjugates to two thermoprecipitating polymers, poly(N‐vinyl caprolactam) and poly(N‐isopropylacrylmide), 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 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 particle of the dispersion of thermoprecipitated ligand‐polymer precipitate may result in stripping of enzyme molecules from the surface of the particles. © 1993 Wiley & Sons, Inc.