A SITE-DIRECTED MUTANT OF CU,ZN-SUPEROXIDE DISMUTASE MODELED AFTER NATIVE EXTRACELLULAR-SUPEROXIDE DISMUTASE

The well-studied cytosolic Cu,Zn-superoxide dismutase (SOD) protects a gainst reperfusion injury although its short (6 min) plasma half-life and negative charge create undesirable pharmacokinetics. We have desig ned, cloned, and expressed a genetic variant of SOD with altered pharm acological properties. A fusion gene consisting of the entire coding r egion of human SOD followed by a positively charged carboxy-terminal ( C-terminal) ''tail'' of eight glycine and six arginine residues was co nstructed. The tail was modeled after the extracellular SOD (EC-SOD) C -terminal 26-amino acid basic peptide. This EC SOD tail binds to hepar in-like proteoglycans on cell surfaces and contributes to the enzyme's very long (30 h) plasma clearance time. After expression in Escherich ia coli, the mutant enzyme was purified and characterized. No differen ces in specific activity or UV absorption spectrum between the mutant and the native enzyme were found. The thermal stability of the fusion protein was greater than that of native SOD. Although native SOD has n o affinity for heparin, the modified enzyme bound to a heparin-agarose column. A ''designer'' SOD able to bind to cell surfaces may aid in t he prevention of superoxide-mediated endothelial damage.