Human extracellular superoxide dismutase is a tetramer composed of two disulphide-linked dimers: a simplified, high-yield purification of extracellular superoxide dismutase.
Studies examining the biochemical characteristics and pharmacological properties of extracellular superoxide dismutase (EC SOD) have been severely limited because of difficulties in purifying the enzyme. Recently EC SOD was found to exist in high concentrations in the arteries of most mammals examined and it is the predominant form of SOD activity in many arteries. We now describe a three-step, high-yield protocol for the purification of EC SOD from human aorta. In the first step, the high affinity of EC SOD for heparin is utilized to obtain a fraction in which EC SOD constitutes roughly 13% of the total protein compared with only 0.3% of that of the starting material. In addition, over 80% of the original EC SOD activity present in the aortic homogenate was retained after the first step of purification. EC SOD was further purified using a combination of cation- and anion-exchange chromatography. The overall yield of EC SOD from this purification procedure was 46%, with over 4 mg of EC SOD obtained from 230 g of aorta. Purified EC SOD was found to exist predominantly as a homotetramer composed of two disulphide-linked dimers. However, EC SOD was also found to form larger multimers when analysed by native PAGE. It was shown by urea denaturation that the formation of multimers increased the thermodynamic stability of the protein. Limited proteolysis of EC SOD suggested that there is one interchain disulphide bond covalently linking two subunits. This disulphide bond involves cysteine-219 and appears to link the heparin-binding domains of the two subunits.
Oury
T D
TD
Department of Pathology, Duke University Medical Center, Durham, NC 27710, USA.
Crapo
J D
JD
Valnickova
Z
Z
Enghild
J J
JJ
D000595:Amino Acid Sequence; D001011:Aorta / Q000201:enzymology*; D004220:Disulfides / Q000737:chemistry; D004795:Enzyme Stability; D006023:Glycoproteins / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D011487:Protein Conformation; D013482:Superoxide Dismutase / Q000737:chemistry*
D004220:Disulfides; D006023:Glycoproteins; D013482:Superoxide Dismutase