Lowry method of protein estimation

Lowry’s assay for total protein is one of the most commonly performed colorimetric assays. This procedure is sensitive because it employs two colour forming reactions. It uses the Biuret reactions in which Cu2+ in presence of a base reacts with a peptide bond of protein under alkaline conditions resulting in reduction of cupric ions (Cu2+) to cuprous ions (Cu+), and Lowry's reaction in which the Folin Ciocaltaeu reagent which contains phosphomolybdic complex which is a mixture of sodium tungstate, sodium molybdate and phosphate, along with copper sulphate solution and the protein, a blue purple colour is produced which can be assessed by measuring the absorbance at 650-700nm.

The blue purple colour is formed due to the reduction of phosphomolybdotungstate to hetero-polymolybdenum blue by the copper catalysed oxidation of aromatic amino acids tryptophan and tyrosine. Thus, the colour intensity depends on the amount of these aromatic amino acids present and will thus vary for different proteins.

The measurement of protein with Folin's reagent has certain advantages. Firstly, it is a sensitive assay which requires no digestion. Secondly, It is 10 or 20 times more sensitive than measurement of the ultraviolet absorption at 280 nm and is much more specific and much less liable to disturbance by turbidities, thirdly, it is several fold more sensitive than the ninhydrin reaction and is simpler, as well as much easier to adapt for small scale analyses. Also it is 100 times more sensitive than the biuret reaction.