Reconstitution of Escherichia coli thioredoxin from complementing peptide fragments obtained by cleavage at methionine-37 or arginine-73.

Abstract Thioredoxin from Escherichia coli (a small hydrogen transport protein containing 108 amino acid residues and having in its oxidized form a single disulfide bond) was acylated with citraconic anhydride. Citraconylation of all amino groups resulted in total loss of enzymatic activity with thioredoxin reductase and immunoprecipitin activity with antithioredoxin antibodies; both these activities were fully restored after deblocking of the citraconylated protein by acid treatment. Large enzymatically inactive peptide fragments of thioredoxin were prepared by selective cleavage at Arg-73 and Met-37, respectively, and tested for their antigenic activity with antibodies against native thioredoxin. Thioredoxin-T-(1-73) and thioredoxin-T-(74-108) were separated by Sephadex G-50 chromatography in 50% acetic acid of a deblocked trypsin digest of citraconylated thioredoxin. Thioredoxin-T-(1-73) afforded about 25% of the corresponding immunoprecipitate of native thioredoxin without significant inhibition at antigen excess. Thioredoxin-T-(74-108) gave no immunoprecipitate but was a potent inhibitor of the reaction of thioredoxin and antithioredoxin as measured by turbidity formation. A major antigenic determinant of thioredoxin was present in the COOH-terminal sequence of the molecule. An equimolar mixture of thioredoxin-T-(1-73) and thioredoxin-T-(74-108) showed full immunoprecipitation activity with antithioredoxin and significant enzymatic activity with thioredoxin reductase. Gel chromatography experiments at pH 8 with the peptide mixture showed a main symmetrical peak with elution volume and amino acid composition identical with native thioredoxin. The results strongly suggested reconstitution of these two fragments to a complex, thioredoxin-T', with a conformation similar to native thioredoxin. Reconstitution of a thioredoxin-like structure was also obtained from a mixture of the overlapping fragments thioredoxin-T-(1-73) and thioredoxin-C-(38-108), although these peptides represented more than the 108 amino acid residues of the protein. Previous results showed reconstitution of thioredoxin-C-(1-37) and thioredoxin-C-(38-108) to a complex called thioredoxin-C' (Holmgren, A. (1972) Fed. Eur. Biochem. Soc. Lett. 24, 351-354). Together with the present results, this shows that three different combinations of two larger peptide fragments obtained by cleavage at two permissible sites gives reconstitution of thioredoxin. In each case, at least one of the component peptides showed strong immunochemical activity with antibodies to native thioredoxin, Netion from a tetrameric to a dimeri