1. Kamran Mukhtar University of Lahore.

2.  The Lowry protein assay is named after Oliver H.  Lowry, who developed and introduced it (Lowry, et al., 1951). It offered a significant improvement over previous protein assays and his paper became one of the most cited references in life science literature for many years.  The Modified Lowry Protein Assay uses a stable reagent that replaces two unstable reagents described by Lowry. Essentially, the assay is an enhanced biuret assay involving copper chelation chemistry.  Although the mechanism of color formation for the Lowry 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 Lowry assay remains poorly understood.  The assay is performed in two distinct steps.  First, protein is reacted with alkaline cupric sulfate in the presence of tartrate for 10 minutes at room temperature. During this incubation, a tetradentate copper complex forms from four peptide bonds and one atom of copper (this is the "biuret reaction").  KAMRAN MUKHTAR

3.  Second, a phosphomolybdic-phosphotungstic acid solution is added. This compound (called Folin-phenol reagent) becomes reduced, producing an intense blue color.  It is believed that the color enhancement occurs when the tetradentate copper complex transfers electrons to the phosphomolybdic-phosphotungstic acid complex.  The blue color continues to intensify during a 30 minute room temperature incubation. It has been suggested that during the 30 minute incubation, a rearrangement of the initial unstable blue complex leads to the stable final blue colored complex which has higher absorbance (Lowry, et al. 1951; Legler, et al. 1985).  The final blue color is optimally measured at 750nm, but it can be measured at any wavelength between 650 nm and 750 nm with little loss of color intensity. It is best to measure the color at 750 nm since few other substances absorb light at that wavelength.  KAMRAN MUKHTAR

4.  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 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 Lowry reagent due to the amino acid interfering with complex formation.  KAMRAN MUKHTAR

5.  Unlike in the BCA assay, the secondary binding step in the Lowry method does not involve detachment of the peptide-copper chelate. Therefore, the Lowry method is effectively an end-point assay.  Although it is always best to include internal standards in any protein assay, it is possible to obtain reasonable protein estimations with this assay method by comparing to a previously-plotted standard curve.  The protocol requires that the Folin phenol reagent be added to each tube precisely at the end of the ten minute incubation.  At the alkaline pH of the Lowry reagent, the Folin phenol reagent is almost immediately inactivated. Therefore, it is best to add the Folin phenol reagent at the precise time while simultaneously mixing each tube. Because this is somewhat cumbersome, some practice is required to obtain consistent results.  KAMRAN MUKHTAR