Biochemical Characterization of G93A Mutant human Cu, Zn Superoxide Dismutase Purified from Transgenic Rat Spinal cord

Abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder with no cure and mutations to Cu‐Zn superoxide dismutase (SOD1) remain the only proven cause of the disease. SOD1, a 32‐kDa homodimer, is a ubiquitously expressed cytosolic antioxidant enzyme with one Cu and one Zn per monomer stabilized by an intra‐subunit disulfide (Cys 57 and Cys 146) bond. It also contains two more cysteines (Cys 6 and Cys 111) thought to be present as reduced thiols. To identify the potential mechanisms by which the mutant SOD1 (mSOD1) mediates neurotoxicity, we purified the mSOD1 from pre‐symptomatic and end stage ALS‐associated G93A mutant rat spinal cords (a model for ALS) using simple reverse‐phase preparative HPLC. The purified mSOD1 was characterized in terms of Cu and Zn levels, SOD activity, and thiol content using chromophoric chelation assay, SOD activity assay and thiol assay respectively. Our results indicate that Zn levels were significantly higher in mSOD1 purified from end stage cord to that of pre‐symptomatic cord, however no significant differences were observed in Cu levels and specific activities. Interestingly, thiol assay revealed that Cys 111 exists primarily in an oxidized state with Cys 6 primarily in the reduced state. These results suggest that biochemical impact of altered Zn affinity and cysteine modifications on SOD1 could have important implications in disease progression.