An efficient method for purification of cuprozinc superoxide dismutase from bovine erythrocytes.
Cuprozinc superoxide dismutase (Cu,Zn-SOD) was isolated from bovine erythrocytes by pH-controlled ammonium sulfate-methanol extraction (ASME extraction). Adjustment of the pH of a suspension of the lysed red cells in the presence of ammonium sulfate (90% saturation) to pH 5.0, followed by partition with an equal amount of methanol, resulted in isolation of the enzyme with specific activity of greater than 2000 units/mg of protein. Further purification using DEAE-cellulose column chromatography gave a highly purified Cu,Zn-SOD showing a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using this procedure about 14 mg of pure Cu,Zn-SOD with a specific activity of 4728 units/mg of protein can be recovered from one liter of bovine blood. The enzyme was characterized and the results obtained were in agreement with earlier reports. This procedure appears, therefore, to be a convenient method for isolating the enzyme.
National Institute for Environmental Studies, Ibaraki, Japan.
Y
Y
Kumagai
M
M
Shinyashiki
G F
GF
Sun
N
N
Shimojo
M
M
Sagai
D000645:Ammonium Sulfate; D000818:Animals; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose; D003300:Copper; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D000432:Methanol; D013482:Superoxide Dismutase / Q000097:blood* / Q000302:isolation & purification*; D015032:Zinc
D003300:Copper; D013482:Superoxide Dismutase; D015032:Zinc; D000645:Ammonium Sulfate; D000432:Methanol