CHARACTERIZATION OF THIOREDOXINS BY SODIUM DODECYL SULFATE-SLAB GEL-ELECTROPHORESIS AND HIGH-PERFORMANCE CAPILLARY ELECTROPHORESIS

Disulfide containing proteins - thioredoxins from E.coli and pig heart mitochondria - were characterized by sodium dodecyl sulfate (SDS)-ele ctrophoresis and high performance capillary electrophoresis (HPCE). Fo llowing the mitochondrial thioredoxin samples at different stages of p urification, we found that their electrophoretic patterns vary, depend ent on the redox condition of isolation, preparation of the samples fo r SDS-electrophoresis, and sample storage. All these factors influence d the relative intensities of several protein bands with thioredoxin-l ike mobility, whereas the sample storage also resulted in the appearan ce of SDS- and dithiothreitol (DTT)-resistant high molecular mass form s, probably thioredoxin dimers. The multiple forms of the thioredoxin from pig heart mitochondria in SDS-electrophoresis might be dependent on the oxidation state of the protein cysteine residues. A commercial preparation of the thioredoxin from E.coli did not exhibit any changes in mobility in SDS gels whether the sample was prepared with or witho ut DTT. After the final purification step no correlation was found bet ween mitochondrial thioredoxin activity, determined in the Insulin ass ay, and its purity in SDS-electrophoresis. A correlation was, however, found when analyzing the thioredoxin by HPCE. The latter approach dem onstrated the heterogeneity of the thioredoxin samples homogeneous on SDS electrophoresis, only one of the several HPCE peaks being active i n the insulin assay. Also, thioredoxin from E.coli, homogeneous on SDS -electrophoresis, was found heterogeneous on HPCE. The peak correspond ing to the insulin-dependent thioredoxin activity was split into two b y DTT treatment, suggesting that redox transformations of thioredoxin could be followed by HPCE.