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)-electrophoresis and high performance capillary electrophoresis (HPCE). Following the mitochondrial thioredoxin samples at different stages of purification, we found that their electrophoretic patterns vary, dependent on the redox condition of isolation, preparation of the samples for SDS-electrophoresis, and sample storage. All these factors influenced the relative intensities of several protein bands with thioredoxin-like mobility, whereas the sample storage also resulted in the appearance of SDS- and dithiothreitol (DTT)-resistant high molecular mass forms, 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 without DTT. After the final purification step no correlation was found between mitochondrial thioredoxin activity, determined in the insulin assay, and its purity in SDS-electrophoresis. A correlation was, however, found when analyzing the thioredoxin by HPCE. The latter approach demonstrated the heterogeneity of the thioredoxin samples homogeneous on SDS electrophoresis, only one of the several HPCE peaks being active in the insulin assay. Also, thioredoxin from E. coli, homogeneous on SDS-electrophoresis, was found heterogeneous on HPCE. The peak corresponding to the insulin-dependent thioredoxin activity was split into two by DTT treatment, suggesting that redox transformations of thioredoxin could be followed by HPCE.
Bunik
V
V
A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia. victoria.bunik@uni-tuebingen.de
Shoubnikova
A
A
Bisswanger
H
H
Follmann
H
H
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004926:Escherichia coli; D012967:Sodium Dodecyl Sulfate; D013552:Swine; D013879:Thioredoxins / Q000737:chemistry*
D001426:Bacterial Proteins; D012967:Sodium Dodecyl Sulfate; D013879:Thioredoxins