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A Biochemistry Laboratory Investigation of Superoxide Dismutase in Bivalves
Author(s) -
Hamilton Mary G
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a299
Subject(s) - hemolymph , chemistry , superoxide dismutase , chromatography , polyacrylamide gel electrophoresis , dismutase , coomassie brilliant blue , biochemistry , fractionation , polyacrylamide , agarose , staining , enzyme , biology , polymer chemistry , genetics
In a small undergraduate college with limited laboratory facilities for teaching the techniques of protein chemistry, we use readily available mollusks, such as bivalves, as a source of interesting, in some cases little studied, enzymes. This semester we concentrated out efforts on superoxide dismutase (SOD). Twelve students (teams of 2) collected the hemolymph, then dissected the gills and mantle and “homogenized” them in a non‐ionic detergent (Pierce). The protein extracts were analyzed by uv‐vis spectroscopy and by colorimetry, and by gel electrophoresis in non‐denaturing systems at pH 8.4 in both non‐sieving agarose and sieving polyacrylamide gels. The denaturing SDS‐PAGE system was used to analyze samples with and without reduction of S‐S bonds. The ability to detect SOD by the staining method of Chen et al. 2001, combined with renaturation with the ion retardation resin (BioRad AG11A8), was key to our investigation. On Western blots, however, we had difficulty correlating the stained activity with the antibody‐labeled bands. For example, in mussel hemolymph, a ca. 90 kDa band of activity was shifted to 37 kDa on reduction, while anti‐ Cu/Zn SOD detected only a “reduced” component with a mass of 58 kDa. Steps have been taken toward isolation of SODs by (NH 4 ) 2 SO 4 fractionation and ion exchange chromatography.