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.
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July 1996
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Research Article|
July 01 1996
Human extracellular superoxide dismutase is a tetramer composed of two disulphide-linked dimers: a simplified, high-yield purification of extracellular superoxide dismutase
Tim D. OURY;
Tim D. OURY
†
*Department of Pathology, Duke University Medical Center, P.O. Box 3712, Durham, NC 27710, U.S.A.
†To whom correspondence should be addressed.
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James D. CRAPO;
James D. CRAPO
*Department of Pathology, Duke University Medical Center, P.O. Box 3712, Durham, NC 27710, U.S.A.
‡Department of Medicine, Duke University Medical Center, P.O. Box 3712, Durham, NC 27710, U.S.A.
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Zuzana VALNICKOVA;
Zuzana VALNICKOVA
*Department of Pathology, Duke University Medical Center, P.O. Box 3712, Durham, NC 27710, U.S.A.
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Jan J. ENGHILD
Jan J. ENGHILD
*Department of Pathology, Duke University Medical Center, P.O. Box 3712, Durham, NC 27710, U.S.A.
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Publisher: Portland Press Ltd
Received:
November 13 1995
Revision Received:
February 07 1996
Accepted:
February 29 1996
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 1996
1996
Biochem J (1996) 317 (1): 51–57.
Article history
Received:
November 13 1995
Revision Received:
February 07 1996
Accepted:
February 29 1996
Citation
Tim D. OURY, James D. CRAPO, Zuzana VALNICKOVA, Jan J. ENGHILD; Human extracellular superoxide dismutase is a tetramer composed of two disulphide-linked dimers: a simplified, high-yield purification of extracellular superoxide dismutase. Biochem J 1 July 1996; 317 (1): 51–57. doi: https://doi.org/10.1042/bj3170051
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