An improved Bathocuproine assay for accurate valence identification and quantification of copper bound by biomolecules

Anal Biochem. 2016 Mar 15;497:27-35. doi: 10.1016/j.ab.2015.12.014. Epub 2015 Dec 30.


Copper is an essential metal in all organisms. Reliably quantifying and identifying the copper content and oxidation state is crucial, since the information is essential to understanding protein structure and function. Chromophoric ligands, such as Bathocuproine (BC) and its water-soluble analog, Bathocuproinedisulfonic acid (BCS), preferentially bind Cu(I) over Cu(II), and therefore have been widely used as optical probes to determine the oxidation state of copper bound by biomolecules. However, the BCS assay is commonly misused, leading to erroneous conclusions regarding the role of copper in biological processes. By measuring the redox potential of Cu(II)-BCS2 and conducting UV-vis absorption measurements in the presence of oxidizable amino acids, the thermodynamic origin of the potential artifacts becomes evident. The BCS assay was improved by introducing a strong Cu(II) chelator EDTA prior to the addition of BCS to prevent interference that might arise from Cu(II) present in the sample. The strong Cu(II) chelator rids of all the potential errors inherent in the conventional BCS assay. Applications of the improved assay to peptides and protein containing oxidizable amino acid residues confirm that free Cu(II) no longer leads to artifacts, thereby resolving issues related to this persistently misused colorimetric assay of Cu(I) in biological systems.

Keywords: BCS assay; Bathocuproine; Biomolecules; Copper; EDTA.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Chelating Agents / chemistry*
  • Copper / analysis*
  • Edetic Acid / chemistry*
  • Electrochemical Techniques
  • Molecular Sequence Data
  • Oxidation-Reduction
  • Peptides / chemistry*
  • Phenanthrolines / chemistry*
  • Proteins / chemistry*
  • Spectrophotometry, Ultraviolet


  • Chelating Agents
  • Peptides
  • Phenanthrolines
  • Proteins
  • Copper
  • Edetic Acid
  • bathocuproine