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.
Greiner
D P
DP
Department of Chemistry, University of California, Davis 95616, USA.
Miyake
R
R
Moran
J K
JK
Jones
A D
AD
Negishi
T
T
Ishihama
A
A
Meares
C F
CF
D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D004492:Edetic Acid / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism; D007501:Iron; D007502:Iron Chelating Agents / Q000138:chemical synthesis*; D009682:Magnetic Resonance Spectroscopy; D009942:Organometallic Compounds / Q000138:chemical synthesis* / Q000378:metabolism; D016339:Spectrometry, Mass, Fast Atom Bombardment
D007502:Iron Chelating Agents; D009942:Organometallic Compounds; C042007:1-(4-bromoacetamidobenzyl)EDTA; D004492:Edetic Acid; D007501:Iron; D003545:Cysteine