Protein Assay
Protein assays determine the amount of protein in an unknown solution. They do this by measuring certain distinctive protein characteristics which involve UV absorbance. Protein assays enable biochemists to characterize one of the two defining properties of enzymes (the other being activity).
Proteins are polymers of amino acids, where amino acid units are joined by peptide bonds which have partial double bond character and absorb light. Proteins also may contain some amino acids with aromatic rings in the side chains. These distinctive features are the most relevant in the measurement of absorbency.
The usual techniques for determining protein concentration depend on using a standard protein to calibrate measurements for accuracy. All protein assay techniques are prone to inaccuracy due to a variety of variables that may affect absorbency (i.e. interfering substances).
The following techniques are generally used in biochemistry. The first, Bioret uses a Cu2+ peptide bond complex, the second – Lowry, uses a heavy metal complex with aromatic amino acids and the third, the Bradford, uses a dye reaction with amino group side chains.
The Biuret method is not highly sensitive but is the most linear because its colour depends on a direct complex between peptide bonds of the protein and Cu(2+) ion.
The Lowry method (named after the scientist who discovered it) is reliant on the presence of aromatic amino acids in the protein. First a peptide bond complex is formed which is then enhanced by a phosphomolybodate complex with the aromatic amino acids.
The Bradford Protein Assay is based on an absorbance shift in a dye known as Coomassie (also known as Brilliant Blue dye, and first described by Volker Neuhoff, a German Scientist, when he named it after a city called Kumasi in Ghana) when bound to arginine and aromatic amino acid residues found in protein, such as Lys. Unlike other protein assays, the Bradford Protein Assay is less sensitive to interference by various chemicals that could exist in protein samples. The assay can also be done in the presence of detergent which makes it useful for determining protein concentrations for membrane proteins.
Web Resources On Protein Assay
Assay Encyclopedia Article
Brad Olson's Protein Assay Resource
Book Resources On Protein AssayAssay of Protein and Polypeptide Hormones by H. van Cauwenberg and Paul Franchimont
Peptide and Protein Drug Analysis by Ronald E. Reid
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