Human liver manganese superoxide dismutase : purification and crystallization, subunit association and sulfhydryl reactivity

• Published in: European journal of biochemistry Vol. 194; no. 3; pp. 713 - 720
• Main Authors: MATSUDA, Y, HIGASHIYAMA, S, KIJIMA, Y, SUZUKI, K, KAWANO, K, AKIYAMA, M, KAWATA, S, TARUI, S, DEUTSCH, H. F, TANIGUCHI, N
• Format: Journal Article
• Language: English
• Published: Oxford Blackwell 27-12-1990
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chromatography, Gel; Crystallization; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Humans; Hydrogen-Ion Concentration; Indicators and Reagents; Liver - enzymology; Molecular Weight; Oxidoreductases; Protein Denaturation; Sulfhydryl Compounds; Superoxide Dismutase - isolation & purification; Superoxide Dismutase - metabolism; Human; Purification; Small angle light scattering; Molecular weight determination; Liver; Thiohydroxyl group; Crystallization; Superoxide dismutase; Physicochemical properties
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1111/j.1432-1033.1990.tb19461.x

Manganese superoxide dismutase (Mn-SOD) has been purified with a high yield (320 mg) from human liver (2 kg) and crystallized. Low-angle laser light scattering of the enzyme has shown that native enzyme is a tetrametic form. Four of the eight cysteine residues in the tetramer reacted with 5,5'-dithiobis(2-nitrobenzoic acid) or with iodoacetamide. The others were only reactive in protein heated with SDS or urea after reduction with dithiothreitol or 2-mercaptoethanol. The reactive sulfhydryl group was found to be located at Cys196 by amino acid sequence analysis of Nbs2-reactive peptides isolated by activated thiol-Sepharose covalent chromatography. Incubation of Mn-SOD in 1% SDS for 2 or 3 days at 25 degrees C or 5 min at 100 degrees C gave material showing two prominent components on polyacrylamide gel electrophoresis in the presence of 0.1% SDS. The major component had a molecular mass of 23 kDa; the other, 25 kDa. Reduction of the protein by dithiothreitol or 2-mercaptoethanol heated in SDS produced only the 25-kDa monomer species. Essentially, no thiol groups were detected in the 23-kDa form, in which two cysteine residues appear to have been oxidized to form an intrasubunit disulfide. This indicates that Cys196 has a reactive sulfhydryl and appears to be a likely candidate for a mixed disulfide formation in vivo.