biuret test

Illustration of Peptide:Cu matrix

Biuret test is used for detecting the presence of peptide bonds. The biuret test relies on the reaction between copper(II) ions and peptide bonds in an alkaline solution. A violet color indicates the presence of proteins. Proteins give a strong biuret reaction because they contain a large number of peptide bonds. It is possible to use the biuret reaction to determine the concentration of proteins because (for most proteins) peptide bonds occur with approximately the same frequency per gram of material. The biuret test works by the peptides forming a matrix by arranging around a Cu²⁺ ion as shown in the illustration to the right. This matrix thus has a pink to violet color depending on the amount of protein.

Procedure

1. Mix 1 cm³ of the sample solution and a few drops of dilute sodium hydroxide or potassium hydroxide solution in a test tube. (It is not compulsory to use sodium hydroxide. Any alkaline substance will work, provided that the alkali is not so strong that it denatures the proteins under test). Shake the tube gently.
2. Add 1% copper(II) sulfate solution drop by drop (Too much copper(II) sulfate would mask the result of the reaction). Shake the test tube after each addition. A violet color indicates the presence of several peptide bonds. It does not indicate the presence of proteins or polypeptides. Other tests must be used to indicate whether the substance testing positive during the biuret test is either a polypeptide and/or a protein.

References

1. Gold. 1990. Organic Compounds in Biological Systems, 2nd ed. John Wiley & Sons, Inc.

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