Thermo Scientific Pierce Protein Assay Technical Handbook Version 2

Modified Lowry Protein Assay
Modified Lowry Protein Assay
Although the mechanism of color formation for the Modified
Lowry Protein Assay is similar to that of the BCA Protein Assay,
there are several significant differences between the two.
In 1951 Oliver H. Lowry introduced this colorimetric total protein
assay method. It offered a significant improvement over previous
protein assays and his paper became one of the most cited references
in the life science literature. The Modified Lowry Protein
Assay uses a stable reagent that replaces two unstable reagents
described by Dr. Lowry. The Modified Lowry assay is easy to
perform because the incubations are done at room temperature
and the assay is sensitive enough to allow the detection of total
protein in the low microgram per milliliter range. Essentially, the
Modified Lowry protein assay is an enhanced biuret assay involving
copper chelation chemistry.
Chemistry of the Modified Lowry Protein Assay
Although the mechanism of color formation for the Modified
Lowry Protein Assay is similar to that of the BCA Protein
Assay, there are several significant differences between the
two. The exact mechanism of color formation in the Modified
Lowry Protein Assay remains poorly understood. It is known that
the color-producing reaction with protein occurs in two distinct
steps. As seen in Figure 1, protein is first reacted with alkaline
cupric sulfate in the presence of tartrate during a 10-minute
incubation at room temperature. During this incubation, a
tetradentate copper complex forms from four peptide bonds and
one atom of copper. The tetradentate copper complex is light
blue in color (this is the “biuret reaction”). Following the incubation,
Folin phenol reagent is added. It is believed that the color
enhancement occurs when the tetradentate copper complex
transfers electrons to the phosphomolybdic/phosphotungstic
acid complex (the Folin phenol reagent).
The reduced phosphomolybdic/phosphotungstic acid complex
produced by this reaction is intensely blue in color. The Folin
phenol reagent loses its reactivity almost immediately upon
addition to the alkaline working reagent/sample solution. The blue
color continues to intensify during a 30-minute room temperature
incubation. It has been suggested by Lowry, et al. and by Legler,
et al. that during the 30-minute incubation, a rearrangement of the
initial unstable blue complex leads to the stable final blue colored
complex that has higher absorbance.
For small peptides, the amount of color increases with the size
of the peptide. The presence of any of five amino acid residues
(tyrosine, tryptophan, cysteine, histidine and asparagine) in the
peptide or protein backbone further enhances the amount of color
produced because they contribute additional reducing equivalents
to further reduce the phosphomolybdic/phosphotungstic
acid complex. With the exception of tyrosine and tryptophan, free
amino acids will not produce a colored product with the Modified
Lowry Reagent; however, most dipeptides can be detected. In the
absence of any of the five amino acids listed above in the peptide
backbone, proteins containing proline residues have a lower color
response with the Modified Lowry Reagent due to the amino acid
interfering with complex formation.
R
R
CH
C NH CH C NH
O
O
Peptide
Bonds
+
Cu 2+
OH –
Tetradentate
Cu 1+
Complex
O
O
CH
R
C NH CH C NH
Protein
Tetradentate
Cu 1+
Complex
+
Mo 6+ /W 6+
OH -
Folin Reagent
(phosphomolybdic/phosphotungstic acid)
BLUE
A max = 750nm
Figure 1. Reaction schematic for the Modified Lowry Protein Assay.
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