Purification of Thioredoxin from Rat Novikoff Ascites Hepatoma

Partial purification of a thioredoxin system from Novikoff ascites hepatoma cells has been previously reported (Moore, E. C. (1967) Biochem. Biophys. Res. Commun. 29, 264–268). Thioredoxin from the same mammalian source has now been purified to electrophoretic homogeneity by ammonium sulfate fractionation, heat treatment, DEAE-cellulose chromatography, and Sephadex chromatography. 1-Dimethylaminonaphthalene-5-sulfonyl determination indicated valine to be the sole NH2-terminal amino acid. Based on a molecular weight of 11,400 estimated by gel filtration, summation of assumed integral numbers of amino acid residue weights was 12,100. Although purification of thioredoxin reductase to electrophoretic homogeneity was not attained in these experiments, a molecular weight value of about 66,700 was estimated by gel filtration. Heterologous cross-reaction between Escherichia coli ribonucleoside diphosphate reductase and Novikoff hepatoma thioredoxin was confirmed but no heterologous cross-reaction between E. coli thioredoxin reductase and Novikoff hepatoma thioredoxin was observed.