Proteins generally absorb UV light at 280 nm while peptide bonds absorb UV light at 214 nm. When quantifying proteins using the Lowry and Buiret methods, absorbance or optical density is measured at 540 nm. Shouldn't absorbance be measured at 280 nm instead of at 540 nm Considering we are measuring absorbance for proteins?
These methods do not measure the absorbance of the protein. The principle of the Lowry assay is described here, taken from the Wikipedia article (https://en.wikipedia.org/wiki/Lowry_protein_assay):
"The method combines the reactions of copper ions with the peptide bonds under alkaline conditions (the Biuret test) with the oxidation of aromatic protein residues. The Lowry method is based on the reaction of Cu+, produced by the oxidation of peptide bonds, with Folin–Ciocalteu reagent (a mixture of phosphotungstic acid and phosphomolybdic acid in the Folin–Ciocalteu reaction). The reaction mechanism is not well understood, but involves reduction of the Folin–Ciocalteu reagent and oxidation of aromatic residues (mainly tryptophan, also tyrosine). Experiments have shown that cysteine is also reactive to the reagent. Therefore, cysteine residues in protein probably also contribute to the absorbance seen in the Lowry assay.[4] The result of this reaction is an intense blue molecule known as heteropolymolybdenum Blue.[5] The concentration of the reduced Folin reagent (heteropolymolybdenum Blue) is measured by absorbance at 660 nm.[6] As a result, the total concentration of protein in the sample can be deduced from the concentration of tryptophan and tyrosine residues that reduce the Folin–Ciocalteu reagent."
We develop colour that has maximum absorbance at 540nm The principle involved in Lowry and Buiret method Colour is based on colour development
At 280nm UV region we measure Absorbance of the protein solutions as such
For lowry or bradford method for protein detection, the reagent will create dark blue color which can be detected at 595nm. But for protein detection without any reagent included, we can use 280nm or 214nm as a wavelength and the best equipment to use is hplc
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