Protein Essay

Chemistry of Protein Assays

Most colorimetric protein assay methods can be divided into two groups based on the type of chemistry involved: those involving protein-copper chelation with secondary detection of the reduced copper and those based on protein-dye binding with direct detection of the color change associated with the bound dye.

  • Copper-based Thermo Scientific Protein Assays:
    • Pierce BCA Protein Assays
    • Pierce Modified Lowry Assay
  • Dye-based Thermo Scientific Protein Assays:
    • Pierce Coomassie (Bradford) Assay
    • Pierce Coomassie Plus (Bradford) Assays
    • Pierce 660 nm Assays

These products are well-characterized, robust assays that provide consistent, reliable results. Nevertheless, each assay reagent has its limitations; having a basic understanding of the chemistries involved with each type of assay is essential for selecting an appropriate method for a given sample and for correctly evaluating results.

Copper-based Assay Chemistries
Dye-based Assay Chemistries

Copper-based Assay Chemistries

Peptides and the Biuret Reaction

Copper-based protein assays, including the BCA and Lowry methods, depend on the well-known “biuret reaction”, whereby peptides containing three or more amino acid residues form a colored chelate complex with cupric ions (Cu2+) in an alkaline environment containing sodium potassium tartrate. This became known as the biuret reaction because it is chemically similar a complex that forms with the organic compound biuret (NH2-CO-NH-CO-NH2) and the cupric ion. Biuret, a product of excess urea and heat, reacts with copper to form a light blue tetradentate complex.

The Biuret Reaction
Diagram of the biuret reaction. By reducing the copper ion from cupric to cuprous form, the reaction produces a faint blue-violet color.
Similarity of biuret and peptide structures
Structures of urea, biuret and peptide. Because polypeptides have a structure similar to biuret, they are able to complex with copper by the biuret reaction.

Single amino acids and dipeptides do not give the biuret reaction, but tripeptides and larger polypeptides or proteins will react to produce the light blue to violet complex that absorbs light at 540nm. One cupric ion forms a colored coordination complex with four to six nearby peptides bonds. The intensity of the color produced is proportional to the number of peptide bonds participating in the reaction. Thus, the biuret reaction is the basis for a simple and rapid colorimetric reagent of the same name for quantitatively determining total protein concentration. The working range for the biuret assay is 5-160mg/mL, which is adequate for some types of industrial applications but not nearly sensitive enough for most protein research needs.

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