Human liver manganese superoxide dismutase

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Abstract
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°C or 5 min at 100°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.