|MadSci Network: General Biology|
The reagent used in the Biuret Test is a solution of copper sulfate (CuSO4) and potassium hydroxide (KOH). The KOH is there to raise the pH of the solution to alkaline levels; the crucial component is the copper (II) ion from the CuSO4.
When peptide bonds are present in this alkaline solution, the copper (II) ions will form a coordination complex with four nitrogen atoms involved in peptide bonds, as described in the figure below.
In this figure, the nitrogens on the left are adjacent in the sequence of one peptide, and the nitrogens on the right are adjacent in the sequence of another peptide (or another section of the same peptide). As you can see, the longer a peptide, the more of these complexes you can form.
Copper Sulfate solution is a blue color, but when the copper (II) ions are coordinated with the nitrogen atoms of these peptide bonds, the color of the solution changes from blue to violet. This color change is dependent on the number of peptide bonds in the solution, so the more protein, the more intense the change. When the peptides are very short, the solution turns a pink color, rather than violet.
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