title: Oxidative polypeptide cleavage mediated by EDTA-Fe covalently linked to cysteine residues.
abstract: Chemical cleavage with reactive oxygen species generated by EPD-Fe, a protein-tethered EDTA-Fe reagent, has been proposed as a method to map the structure of nonnative equilibrium protein folding intermediates [Ermácora, M. R., Delfino, J. M., Cuenoud, B., Schepartz, A., & Fox, R. O. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6383-6387]. The chemical structure of protein cleavage products and the mechanism of backbone scission for this class of reagents have been unclear. Here, we report the nature of EPD-Fe-mediated backbone cleavage of a small model peptide. The EPD-Fe reagent was attached to a partially alpha-helical peptide, alpha 1BA1a (Ac-AEAEEAAKKAKEACKA-NH2), through a mixed disulfide. Backbone cleavage was initiated by addition of the iron reductant ascorbate. Chemical analysis of the novel cleavage products revealed an oxidative cleavage mechanism, probably initiated by diffusible hydroxyl radicals. The EPD-Fe-mediated cleavage technique appears to be suitable for the analysis of nonnative protein states such as the molten globule.
authors.affiliation: Howard Hughes Medical Institute, Yale University, New Haven, Connecticut.
authors.forename: I E
authors.initials: IE
authors.lastname: Platis
authors.forename: M R
authors.initials: MR
authors.lastname: Ermácora
authors.forename: R O
authors.initials: RO
authors.lastname: Fox
mesh_terms: D000595:Amino Acid Sequence; D001205:Ascorbic Acid / Q000737:chemistry; D002942:Circular Dichroism; D004492:Edetic Acid / Q000031:analogs & derivatives* / Q000737:chemistry*; D005290:Ferric Compounds / Q000737:chemistry*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D011487:Protein Conformation; D017382:Reactive Oxygen Species / Q000737:chemistry
chemical_list: C084719:EDTA-(2-aminoethyl 2-pyridyl disulfide)-Fe; D005290:Ferric Compounds; D010446:Peptide Fragments; D010455:Peptides; D017382:Reactive Oxygen Species; C084718:alpha1BAla; D004492:Edetic Acid; C019179:Fe(III)-EDTA; D001205:Ascorbic Acid