(607d) Synthetic Zwitterlation of Enzymatic Proteins for Stability and Retained Enzymatic Kinetics

A poly(zwitterion)-protein conjugate has been made to provide protein stability in human serum, high concentration urea, and at high temperatures. A new synthetic method has been developed to produce a N-hydroxysuccinimide terminated zwitterionic polymer for use in protein conjugation. Good control and PDI's are shown due to its synthesis in organic solvents followed by post modification into its zwitterionic structure. Alpha-chymotrypsin was chosen as a model enzyme for conjugation to the zwitterionic polymer, poly(carboxybetaine) (pCB). Poly(ethylene glycol) (PEG), a well known and widely used protein stabilizing polymer, was used as a comparison. We have shown that not only do pCB enzyme conjugates posses show stability under the tested conditions, but do so without adjusting their kinetics. Polymer density was also adjusted (polymers/protein) and shown how higher density polymers provide increased stability with both polymers. Commonly, as seen with PEG, polymer inhibition is popularly known for increasing the dissociation constant of the enzyme substrate complex. We hypothesize that this is due to the presence of the highly solvated polymer preventing diffusion of substrate into the active site. pCB, as a poly(osmolite) of betaine, interacts more with the protein surface and less with the surrounding water, therefore stabilizing the structure of the protein, without inhibiting substrate diffusion. This provides evidence for use of pCB polymers to provide stability to protein conjugates for use in the biomedical industry. This provides evidence for using pCB as a more effective polymer conjugate for enzyme and receptor binding proteins.