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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

Takaaki Akaike

Keizo Sato

Hiroshi Maeda

Department of Microbiology, Kumamoto University School of Medicine, Kumamoto 860, Japan

Takashi Hirano

National Institute of Bioscience and Human Technology, 1-1 Tsukuba-Higashi, Ibaraki 305, Japan

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.

Journal of Bioactive and Compatible Polymers, Vol. 11, No. 3, 169-190 (1996)
DOI: 10.1177/088391159601100301


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