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.