Purification and characterization of a Cu,Zn-SOD from garlic (Allium sativum L.). Antioxidant effect on tumoral cell lines.
Crude garlic extract contains one Mn-superoxide dismutase designated as SOD1 and two Cu,Zn superoxide dismutases as SOD2 and SOD3. The major isoform SOD2 was purified to homogeneity by Sephacryl S200-HR gel filtration, DEAE Sepharose ion exchange chromatography, and chromatofocusing using PBE 94. SOD2 was purified 82-fold with a specific activity of 4,960 U/mg protein. This enzyme was stable in a broad pH range from 5.0 to 10.0 and at various temperatures from 25 to 60 degrees C. The native molecular mass of SOD2 estimated by high performance liquid chromatography on TSK gel G2000SW column was 39 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed a single band near 18 kDa, suggesting that native enzyme was homodimeric. The isoelectric point as determined by chromatofocusing was 5. Analysis of its N terminal amino acid sequence revealed high sequence homology with several other cytosolic Cu,Zn-SODs from plants. Exposure of cancer cell lines to garlic Cu,Zn-SOD2 led to a significant decrease in superoxide content with a concomitant rise in intracellular peroxides, indicating that the enzyme is active in mammalian cells and could, therefore, be used in pharmacological applications.
Hadji
Imen
I
National Institute of Applied Sciences and Technology, Tunis Cedex, Tunisia.
Marzouki
M Nejib
MN
Ferraro
Daniela
D
Fasano
Elena
E
Majdoub
Hafedh
H
Pani
Giovanni
G
Limam
Ferid
F
D000818:Animals; D000975:Antioxidants / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D045744:Cell Line, Tumor; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005737:Garlic / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D051379:Mice; D018384:Oxidative Stress / Q000187:drug effects; D013482:Superoxide Dismutase / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D013481:Superoxides / Q000037:antagonists & inhibitors / Q000378:metabolism; D013696:Temperature
D000975:Antioxidants; D007527:Isoenzymes; D013481:Superoxides; D013482:Superoxide Dismutase