Characterization of the heterogeneity of polyethylene glycol-modified superoxide dismutase by chromatographic and electrophoretic techniques.

Covalent attachment of polyethylene glycol (PEG) chains to the enzyme Cu,Zn-superoxide dismutase (SOD) produces a heterogeneous mixture of modified protein species. The heterogeneity of the product (PEG-SOD) derives from a variable stoichiometric combination of PEG with individual SOD molecules in addition to the polydispersity of the PEG reagent. Characterization of PEG-SOD presents significant challenges due in part to this heterogeneity in addition to the hybrid nature of the modified enzyme. The application of classical methods of protein characterization is not always successful for these PEG-proteins requiring the development of alternative or modified procedures. A series of chromatographic techniques including reversed-phase, ion-exchange, size-exclusion, and hydrophobic interaction high-performance liquid chromatography along with electrophoretic techniques including isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and capillary zone electrophoresis have been developed for assessing the degree of heterogeneity of PEG-SOD samples which encompass a range of different stoichiometries. Examples will be given demonstrating the application of these techniques to characterize PEG-SOD samples of different composition produced during the course of the reaction between SOD and an activated PEG reagent.