Synthesis and characterization of bioaffinity interactive heterobifunctional polyethylene glycols

Abstract

Novel methods for synthesis of monoprotected homobifunctional polyethylene glycols and subsequent synthesis and application of heterobifunctional derivatives in protein immobilization and separation is described. In addition, modification of proteins with PEG and properties of the conjugates are presented. Heterobifunctional PEG derivatives having a chelate at one end and at the other one specific ligands for proteins have been synthesized. Metal binding constants and kinetic parameters of these bifunctional chelate polymers were found in excellent correlation with the binding affinities shown by corresponding unconjugated groups. These heterobifunctional PEG derivatives were used to characterize the partitioning behavior of several proteins in Affinity Two-Phase Partitioning, ATPP, at different conditions. Both sides of these bioaffinity chelating polymers were found to be effective in the partitioning of these model proteins. These modified heterobifunctional affinity chelating polymers were also adsorbed in aqueous media to different chelating adsorbents used in IMAC separation. In this scheme an IMA-adsorbent could be transformed to a more selective affinity separation mode, by adding an affinity-chelating ligand. The attached ligand could be removed by weakening the metal interactions between the two chelates in the system which allow the column to operate again in the original IMAC mode, if desired. The amount of these bioligands bound to the columns were a function of the type of the IMA-adsorbents, pH, salts and the metal immobilized on the gels. Trypsin and avidin were bound on columns loaded with a PAB-PEG-chelate and a biotin-PEG-chelate respectively. As a typical example, bound trypsin was eluted from the columns with the trypsin inhibitor, benzamidine, acting as a competitive ligand. The bioligands were eluted reversibly from the IMA-adsorbents, using free IDA as a competitive ligand, using low pH buffers or EDTA. PEG derivatives of 5000 daltons, were chemically fixed to non essential groups on trypsin through amidation with amino groups using two different methods. Kinetic studies were performed upon modification to determine the activity and stability of the modified biomolecule under these conditions. In both cases the modified enzyme adduct, retained their original biological activities and showed substantial changes in properties.