Analysis of polyethylene glycol modified superoxide dismutase by chromatographic, electrophoretic, light scattering, chemical and enzymatic methods.

Abstract

Covalent conjugation of bovine erythrocyte superoxide dismutase (SOD) with activated polyethylene glycol (PEG) results in a mixture of modified species (PEG-SOD) with properties different from those of the native enzyme. The components of this mixture were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing, and chromatographic (size-exclusion, anion-exchange, cation-exchange and reverse-phase high performance liquid chromatography) techniques. Physicochemical properties such as apparent molecular weight, isoelectric point, relative hydrophobicity and relative cation-anion charge number were measured by electrophoretic and chromatographic procedures. Dispersity and apparent radius were examined by chromatographic and light scattering techniques. The extent of covalent modification and enzymatic activity change were measured by chemical and spectroscopic methods, showing that activity loss was not due to catalytic site modification. The properties of the PEG-modified form of the enzyme were compared with those of native SOD and showed that in addition to changing biological properties, PEG modification of proteins can result in a product with unexpectedly high heterogeneity and substantial changes in isoelectric point and hydrophobicity. Altered biological properties may therefore not merely be due to shielding of protein surface by PEG chains. Apparent properties of PEG modified proteins such as molecular weight were found to be highly method dependent, with poor agreement being shown among classical measurements.