A Specific Absorbance to Estimate a Protein by Lowry’s Method

Lowry’s assay is the most common colorimetric method to determine protein content in the solution. Under alkaline conditions, cupric ions (Cu2+) chelate with nitrogen atoms of the peptide bonds with the consequence of a reduction of cupric (Cu2+) to cuprous ions (Cu+). The cuprous ions (Cu+) are reduced by folin’s reagent (phosphomolybdic/phosphotungstic acid) to form tungsten blue. This study is receptive to reducing agents and other routine lab reagents. Here, it was aimed to estimate the protein content by Lowry’s method at different wavelengths by UV-visible spectrophotometric analysis. Using Bovine Serum Albumin (BSA) particularly as a standard protein estimation of proteins is done because of its low price, high purity and ready availability. The concentration of albumin used in the range of 20–200 μg/ml. By different aliquots of BSA with known concentration, the unknown concentration of the sample was measured spectrophotometrically at 540 nm, 680 nm and 720 nm. The intensity of the blue colour formed is directly proportional to the protein concentration of the solution. Plotting concentration of standard protein Vs absorbance has shown the linearity in all 3 different wavelengths i.e., 540 nm, 680 nm, and 720 nm in which the line through all points was at 540 nm than at 680 nm or 720 nm. Either use of higher concentration of the standard solution or higher range of wavelength can lead to the biphasic graph, which shows ambiguity in the study. Therefore, at a wavelength of 540 nm can be a specific absorbance to measure a protein in unknown solution. By these, most of the biochemical studies that involve the measurement the protein content in different solutions and the specific activity of a particular enzymatic activity have shown some importance when proteins exist in purified form or unlike samples are being studied with the Lowry’s method.