Document Detail


Development of a methodology based on metal-catalyzed oxidation reactions and mass spectrometry to determine the metal binding sites in copper metalloproteins.
MedLine Citation:
PMID:  12641237     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Efforts have been made to develop a method that uses metal-catalyzed oxidation (MCO) reactions and mass spectrometry (MS) to identify the binding site of copper in metalloproteins. This method uses MCO reactions to oxidize the amino acids in the metal-binding site and MS to identify the amino acids that have been oxidized. Several reaction conditions, including Cu(II)/ascorbate/O2, Cu(II)/O2/H2O2, and Cu(II)/ascorbate/O2/H2O2, have been tested at varying concentrations to find the optimum conditions for specific oxidation of only the amino acids bound to copper. For small peptides, such as angiotensin I (Agt I) and [Gln11]-amyloid-beta-protein fragment 1-16 (A beta(1-16)), the optimum conditions for specific modification involve the use of Cu(II)/ascorbate/O2. For a larger protein, azurin, the speed and specificity of the MCO reactions are enhanced by the presence of a relatively high concentration of ascorbate (100 mM) and a small concentration of H2O2 (1 mM). Optimized reaction conditions combined with MS/MS and MSn analysis on a quadrupole ion trap mass spectrometer allow the copper-binding sites to be specifically identified. For Agt I and A beta(1-16), the amino acids bound to copper can be identified without any false positives. For azurin, four of the five amino acids bound to copper are identified with one false positive. This false positive, however, corresponds to the oxidation of Met44, which is probably due to its susceptibility to oxidation and its proximity to the only residue not identified (i.e., Gly45). The results altogether suggest that MCO reactions and MS provide a very promising approach for identifying the amino acid residues bound to copper in metalloproteins.
Authors:
Jihyeon Lim; Richard W Vachet
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Analytical chemistry     Volume:  75     ISSN:  0003-2700     ISO Abbreviation:  Anal. Chem.     Publication Date:  2003 Mar 
Date Detail:
Created Date:  2003-03-18     Completed Date:  2004-04-13     Revised Date:  2006-11-15    
Medline Journal Info:
Nlm Unique ID:  0370536     Medline TA:  Anal Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  1164-72     Citation Subset:  IM    
Affiliation:
Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, USA.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Angiotensin I / chemistry
Binding Sites
Catalysis
Copper / chemistry*
Humans
Mass Spectrometry
Metalloproteins / chemistry*
Metals / chemistry*
Molecular Sequence Data
Oxidation-Reduction
Chemical
Reg. No./Substance:
0/Metalloproteins; 0/Metals; 7440-50-8/Copper; 9041-90-1/Angiotensin I

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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