Polymer Conjugation to Cu,Zn-SOD and Suppression of Hydroxyl Radical Generation on Exposure to H2O2: Improved Stability of SOD in Vitro and in Vivo

Abstract

Highly reactive hydroxyl radical (OH) is generated during reaction of Cu,Zn-SOD (SOD)1 with H202, its own enzymatic reaction product (SOD plus 0;-). During inactivation of Cu,Zn-SOD with H202, Cu2+ liberated from the SOD catalyzes the formation of OH from H202. The OH thus generated will react with a,-protease inhibitor (a,-PI) if it is available in the milieu. To circumvent OH generation, we prepared eight different polymer-SOD conjugates and compared their stability against H202 treatment. Furthermore, the effect of each polymer-SOD conjugate on a,-PI in the presence of H202 was tested. Most polymer-SOD conjugates, except poly(styrene-co-maleic acid butylate; SMA)-SOD and poly(ethylene glycol; PEG)-SOD, showed higher stability than native SOD against inactivation by H202. The residual activity of divinylether maleic acid/anhydride copolymer (pyran copolymer)-conjugated SOD was 87% of the initial activity after incubation with 0.1 mM H202 for 2 h, whereas that of native SOD was 49%. Antiprotease activity of ca,-PI was completely abrogated when the inhibitor was treated with native SOD and 3.0 mM H202, but there was no change with pyran copolymer-SOD after 3 h of incubation with H202. Our results suggest that pyran copolymer captured loosely bound Cu2+ during the reaction of SOD with H202, thus resulting in suppression of both 0OH-mediated inactivation of SOD and suppression of activity of a,-PI. PEG showed little Cu2+-binding activity; no appreciable protection of SOD and a,-PI against OH generation was observed when Cu,Zn-SOD was conjugated with PEG. Circular dichroism and electron spin resonance studies of Cu,Zn-SOD treated with H202 indicated that the peptide conformation as well as the copper ligand of Cu,Zn-SOD can be stabilized not only by pyran copolymer per se but also by conjugation of Cu,Zn-SOD with some copolymers such as pyran.