Enzymic Reduction of Disulfide Bonds by Thioredoxin

The NADPH‐dependent enzymic reduction of disulfide bonds in human choriogonadotropin and its two subunits, α and β, was examined with thioredoxin and thioredoxin reductase from Escherichia coli. With 12μM thioredoxin and 0.1 μM thioredoxin reductase at pH 7 all disulfide bonds in the α subunit could be reduced in 15 min. The reduction of disulfide bonds was recorded by a simple spectrophotometric assay at 340 nm, which allowed quantitation of the reduction rate and the number of disulfide bonds reduced. Partial reduction of the α subunit with thioredoxin followed by S‐carboxymethylation with iodo[2‐ 3 H]acetic acid and analysis of tryptic peptides indicated that all S‐S bonds in the a subunit were surface oriented and equally reactive. The usefulness of thioredoxin reducide bonds as a chemical probe of protein structure was shown by the much slower reaction of disulfide bonds in the intact hormone as compared to its two biologically inactive subunits.