Superoxide dismutase and catalase conjugated to polyethylene glycol increases endothelial enzyme activity and oxidant resistance.
Covalent conjugation of superoxide dismutase and catalase with polyethylene glycol (PEG) increases the circulatory half-lives of these enzymes from less than 10 min to 40 h, reduces immunogenicity, and decreases sensitivity to proteolysis. Because PEG has surface active properties and can induce cell fusion, we hypothesized that PEG conjugation could enhance cell binding and association of normally membrane-impermeable enzymes. Incubation of cultured porcine aortic endothelial cells with 125I-PEG-catalase or 125I-PEG-superoxide dismutase produced a linear, concentration-dependent increase in cellular enzyme activity and radioactivity. Fluorescently labeled PEG-superoxide dismutase incubated with endothelial cells showed a vesicular localization. Mechanical injury to cell monolayers, which is known to stimulate endocytosis, further increased the uptake of fluorescent PEG-superoxide dismutase. Endothelial cell cultures incubated with PEG-superoxide dismutase and PEG-catalase for 24 h and then extensively washed were protected from the damaging effects of reactive oxygen species derived from exogenous xanthine oxidase as judged by two criteria: decreased release of intracellular 51Cr-labeled proteins and free radical-induced changes in membrane fluidity, measured by electron paramagnetic resonance spectroscopy of endothelial membrane proteins covalently labeled with 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl. Addition of PEG and PEG-conjugated enzymes perturbed the spin-label binding environment, indicative of producing an increase in plasma membrane fluidity. Thus, PEG conjugation to superoxide dismutase and catalase enhances cell association of these enzymes in a manner which increases cellular enzyme activities and provides prolonged protection from partially reduced oxygen species.
D000818:Animals; D001013:Aorta, Thoracic / Q000201:enzymology; D001692:Biological Transport; D002374:Catalase / Q000378:metabolism*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000201:enzymology*; D004795:Enzyme Stability; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007700:Kinetics; D010084:Oxidation-Reduction; D011092:Polyethylene Glycols; D013482:Superoxide Dismutase / Q000378:metabolism*; D013552:Swine; D013861:Thiocyanates
D005452:Fluoresceins; D005456:Fluorescent Dyes; D013861:Thiocyanates; D011092:Polyethylene Glycols; D002374:Catalase; D013482:Superoxide Dismutase; D016650:Fluorescein-5-isothiocyanate