OXIDATIVE POLYPEPTIDE CLEAVAGE MEDIATED BY EDTA-FE COVALENTLY-LINKED TO CYSTEINE RESIDUES

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 [Ermacora, M. R., Delfino, J. M., Cuenoud, B., Schepartz, A., & Fox, R . 0. (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 pept ide. The EPD-Fe reagent was attached to a partially alpha-helical pept ide, alpha1 BAla (Ac-AEAEEAAKKAKEACKA-NH2), through a mixed disulfide. Backbone cleavage was initiated by addition of the iron reductant asc orbate. Chemical analysis of the novel cleavage products revealed an o xidative cleavage mechanism, probably initiated by diffusible hydroxyl radicals. The EPD-Fe-mediated cleavage technique appears to be suitab le for the analysis of nonnative protein states such as the molten glo bule.