Some properties of polyethylene glycol:phenylalanine ammonia-lyase adducts

Abstract

Methoxypolyethylene glycol of 5000 daltons (PEG) was attached covalently to phenylalanine ammonia-lyase from Rhodotorula glutinis. Attachment of sufficient quantities of PEG to phenylalanine ammonia-lyase substantially reduces immunological recognition and clearance of the conjugated enzyme in mice. The modified enzyme demonstrates altered catalytic properties such as shifts in the pH and temperature optima, an increase in the Michaelis-Menten constant, and a lowered Vmax in comparison with the native enzyme. PEG-phenylalanine ammonia-lyase has increased resistance to proteolytic digestion, particularly when in the presence of cinnamate, a competitive inhibitor, while the native enzyme is rapidly inactivated. In the ultracentrifuge PEG-phenylalanine ammonia-lyase exhibits a lower sedimentation rate than the unmodified enzyme, despite the fact that it is much larger. The electrophoretic mobility of PEG-phenylalanine ammonia-lyase is greatly decreased in comparison to the unmodified enzyme. PEG-phenylalanine ammonia-lyase had a much longer blood-circulating life in mice, both initially and after a number of injections, than did the native enzyme. PEG-phenylalanine ammonia-lyase was a good immunogen but a poor antigen in mice and rabbits, that is, it readily induced antibody formation, but reacted poorly in vitro with the antibodies that were formed against it.