Synthesis of the Protein Cutting Reagent Iron (S)-1-(p-Bromoacetamidobenzyl)ethylenediaminetetraacetate and Conjugation to Cysteine Side Chains

Convenient methodology for preparation and conjugation of the protein-cutting iron chelate iron (S)-1-(p-bromoacetamidobenzyl) ethylenediaminetetraacetate (Fe-BABE) is given. This formulation of the reagent can be handled in a manner analogous to many other protein-labeling reagents, such as fluorescent probes or cross-linkers. By taking advantage of the recently discovered peptide hydrolysis reaction, the chelate may be tethered to a single site (e.g., a cysteine side chain) and used to map its proximity to individual peptide bonds by automated Edman sequencing of the protein fragments produced. The method is illustrated by conjugation of Fe-BABE to the carboxy terminal domain (amino acid residues 234-329) of the Escherichia coli RNA polymerase alpha subunit. The molecular mass of the protein conjugate was confirmed by electrospray ionization mass spectrometry.