Effect of covalent attachment of polyethylene glycol on immunogenicity and circulating life of bovine liver catalase.

TLDR

Methoxypolyethylene glycols (PEG‑1900 and PEG‑5000) were covalently attached to bovine liver catalase using 2,4,6‑trichloro‑s‑triazine, and rabbits were immunized intravenously or intramuscularly with the PEG‑catalase preparations (≈43–40 % PEGylation). Intravenous immunization with PEG‑catalase elicited no detectable antibodies, whereas intramuscular immunization produced antibodies to both PEG‑catalase and native catalase; PEG‑1900‑catalase retained 93 % activity, PEG‑5000‑catalase 95 %, resisted protease digestion, and extended circulating half‑life in acatalasemic mice without provoking immune responses or organ damage.

Abstract

Methoxypolyethylene glycols of 1900 daltons (PEG-1900) or 5000 daltons (PEG-5000) were covalently attached to bovine liver catalase using 2,4,6-trichloro-s-triazine as the coupling agent. Rabbits were immunized by the intravenous and intramuscular routes with catalase modified by covalent attachment of PEG-1900 to 43% of the amino groups (PEG-1900-catalase). The intravenous antiserum did not yield detectable antibodies against PEG-1900-catalase or native catalase, as determined by Ouchterlony and complement fixation methods, whereas the intramuscular antiserum contained antibodies to both PEG-1900-catalase and catalase. PEG-1900 did not react with either antiserum. Catalase was prepared in which PEG-5000 was attached to 40% of the amino groups (PEG-5000-catalase). This catalase preparation did not react with either antiserum. PEG-1900-catalase retained 93% of its enzymatic activity; PEG-5000-catalase retained 95%. PEG-5000-catalase resisted digestion by trypsin, chymotrypsin, and a protease from Streptomyces griseus. PEG-1900-catalase and PEG-5000-catalase exhibited enhanced circulating lives in the blood of acatalasemic mice during repetitive intravenous injections. No evidence was seen of an immune response to injections of the modified enzymes. Mice injected repetitively with PEG-5000-catalase remained immune competent for unmodieied catalase, and no evidence of tissue or organ damage was seen.