title: A pulse--radiolysis approach to fast reductive cleavage of a disulfide bond to uncage enzyme activity.
abstract: The essential thiol of the enzyme papain has been caged by linking to an aromatic thiol. The resulting caged protein is inactive but enzymatic activity is fully restored upon chemical cleavage of the protective disulfide bond. We have exploited the chemistry of this disulfide bond to uncage papain by pulse radiolysis. We have shown that up to 10% of the enzyme activity can be restored by reductive pulse radiolysis. This approach has been tested on a small-molecule model system, and experiments on this model compound show that pulse radiolysis of the mixed cysteine-aromatic disulfide results in selective reduction of the disulfide bond to generate a thiol in 10-20% yield, consistent with the radiolytically restored activity of the caged papain quantified by the biochemical assay.
authors.affiliation: Centre for Chemical Biology, Department of Chemistry, Krebs Institute for Biomolecular Science, University of Sheffield, Sheffield S3 7HF, UK. l.milanesi@mail.cryst.bbk.ac.uk
authors.forename: Lilia
authors.initials: L
authors.lastname: Milanesi
authors.forename: Salvador
authors.initials: S
authors.lastname: Tomas
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Hunter
authors.forename: Julia A
authors.initials: JA
authors.lastname: Weinstein
authors.forename: Ruth
authors.initials: R
authors.lastname: Edge
authors.forename: Suppiah
authors.initials: S
authors.lastname: Navaratnam
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Waltho
authors.forename: Jonathan
authors.initials: J
authors.lastname: Best