Characterization of in vitro oxidized barstar.

The polypeptide inhibitor of the ribonuclease barnase, barstar, has two cysteine residues in positions 40 and 82. These have been proposed to form a disulfide bridge leading to an increase in stability without changing the inhibitory activity of the protein. Barstar and a mutant (E80A) were oxidized in vitro and the biochemical and physico-chemical properties of the oxidized monomers were analysed. The oxidized proteins show no inhibition of barnase using a plate assay and are significantly destabilized. CD spectra indicate a loss of secondary structure. The amino acid substitution E80 --> A stabilizes the oxidized barstar to about the same extent as it does the reduced protein, indicating, however, that the helical region which it is in is intact.