Site-specific cleavage of proteins with metal chelates is an approach for designing artificial proteolytic reagents that are directed by proximity to a peptide bond rather than by an amino acid residue type. In the presence of ascorbate and H2O2, an iron chelate attached to Cys-212 of the enzyme human carbonic anhydrase I quickly cleaved the protein between residues Leu-189 and Asp-190 to produce two discrete fragments. The transfer of an 18O atom from [18O]H2O2 (or [18O]O2) to the carboxyl group of Leu-189 was demonstrated by mass spectrometry. Quantitative experiments revealed that one molecule of H2O2 and one molecule of ascorbate afforded the hydrolysis of one peptide bond (1:1:1 stoichiometry) and that the reaction required ascorbate and H2O2. The process is catalytic, since related experiments on the protein bovine serum albumin revealed two cleavage events for each poly peptide chain cleaved. Hydroxyl radical scavengers had no significant effect. These results may be explained by generation of a highly nucleophilic oxygen species, such as peroxide coordinated to the iron chelate, that attacks a carbonyl carbon nearby.
|Original language||English (US)|
|Number of pages||5|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|State||Published - 1991|
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