On which principle does the biuret method operate?

The biuret method operates on the principle of coordinate bonds forming between Cu^2+ ions and the carbonyl and imine groups in peptide bonds of proteins. When proteins are present in a solution containing copper sulfate, the copper ions interact specifically with the peptide bonds that link amino acids together in the protein structure. This interaction results in the formation of a violet-colored complex. The intensity of this color is directly proportional to the protein concentration in the solution, allowing for the quantitative analysis of proteins.

In contrast, other methods listed do not pertain to the biuret test. The first option relates to a different reaction involving phenolic compounds, which is not characteristic of the biuret method. The third option refers to the protein error of indicators, which is relevant in colorimetric assays of proteins but is distinct from the biuret principle. The fourth option involves phosphomolybdic acid, a compound used in other types of assays such as the Kjeldahl method for nitrogen determination, rather than the specific reactions utilized in the biuret method. Thus, the biuret method uniquely depends on the formation of coordinate bonds with copper ions and peptide bonds to assess protein concentration.