What does Biuret test for ? Its Principle, Mechanism and Uses

A biuret test is a chemical assay that helps check for the presence of protein in a given sample. To confirm the presence of protein, it will rely on the changes in color.

An indicator that protein is present is when the color changes to violet. Although the test is called biuret, it does not use the chemical biuret; a substance derived from urea. In fact, biuret is not a protein. However, it has the ability to generate a positive result in the biuret test. (1, 2)

biuret test for protein is distinguished by changes in the color of the mixture from blue to purple

Image 1: A biuret test for protein is distinguished by changes in the color of the mixture from blue to purple.

Picture Source: onlinebiologynotes.com

Why does biuret turn purple in the presence of protein?

Biuret is a product of heating urea to about 180 degrees Celsius. If in alkaline condition, biuret is treated using diluted copper sulfate, the color changes to purple. Hence, it is used to identify proteins in a particular sample, specifically biological fluids. (1, 2, and 3)

Performing a Biuret Test

requirements when performing biuret testImage 2: These are the requirements when performing biuret test.

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Requirements:

  1. Biuret reagent
  2. Water bath
  3. Dry test tubes
  4. Pipettes
  5. 1% alanine and 5% egg white

 

Follow these steps:

  1. Put at least 1 ml of test solutions in the test tube (dry) and another 1 ml in a separate test tube containing 1 mL distilled water as a control.
  2. Put at least 1 mL of biuret reagent in all test tubes.
  3. Watch for any changes in color, specifically blue color. (4, 5, and 6)

 

Two test tubes were subjected to biuret test

Image 3: Two test tubes were subjected to biuret test. The other is heated, which changes the color of the mixture into violet. The unheated mixture remains blue in color.

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What is biuret reagent?

Biuret test uses a reagent consists of potassium hydroxide and copper sulfate. Under normal condition, the color of the biuret reagent is blue. However, it changes its color to violet if peptide bonds are present. The peptide bond is the chemical bond that holds amino acids together. There are other alternatives for biuret reagent and these are copper sulphate and sodium hydroxide. Fehling’s solutions A and B can be used too.

The hydrated copper sulphate is the one responsible for the formation of Cu (II) ions forming the chelate complex. The distinct blue color of the reagent is caused by Cu (II) ions. On the other hand, potassium hydroxide solution is the one responsible for providing the alkaline medium. Lastly, the potassium sodium tartrate causes stabilization of the chelate complex. (3, 4, 5, 6, and 7)

What to keep in mind?

  • If the specimen to be tested is solid, you need to dissolve or liquefy it first.
  • Avoid shaking the mixture.
  • Measure the changes in color using spectrophotometer. This is to assess how much protein is present in a given sample.
  • There is a better way of checking for the protein content in a particular sample. It is the modified biuret test called Smith Assay. It increases the sensitivity of the biuret test by a hundredfold. It is different from the biuret test because it uses bicinchoninic acid as a copper source. When protein is present, it will immediately change its color to purple.
  • The level of protein in the urine or blood has nothing to do with a high protein diet.
  • Biuret test results can be affected by the presence of magnesium and ammonium ions. In a case like this, you need to use excess alkali. (5, 7, 8, and 9)

How to perform the biuret test for protein when using Fehling’s A and B solutions?

  1. Make sure you prepare a fresh Fehling’s A and B solutions.  Keep in mind that A is copper (II) solution while B contains both the solutions of sodium hydroxide and sodium potassium tartrate.
  2. When testing a food sample, you need to add about 1 cm3 of solutions A and B to the specimen.
  3. Repeat the aforementioned steps with de-ionized water to have a negative control. For positive control, you should use albumin or egg white.
  4. Carefully shake the mixture and let it stand there for five minutes.
  5. Watch for any changes in color.  (2, 7, 9, and 10)

Performing biuret test using copper sulphate and sodium hydroxide solutions

  1. Food samples are added with sodium hydroxide solution (1 cm3) and copper (II) sulphate solution (1%).
  2. Follow the steps mentioned above but this time with de-ionized water to create a negative control. The other one is for positive control using albumin or egg white.
  3. Shake the mixture and let it stand for about five minutes.
  4. Carefully observe for any changes in color.

Why is it important to detect the presence of protein in a given type of fluid? 

Detecting the presence of protein in urine is a perfect way to check for illnesses and diseases. For an instance, protein in human urine, especially more than the normal limit may indicate kidney-related problems. It may also indicate other health conditions such as:

  • Diabetes mellitus
  • Leukemia
  • High blood pressure
  • Lupus
  • Pregnancy

On the other hand, a high level of protein in the blood may indicate the following conditions:

  • Infection
  • Dehydration
  • Chronic inflammation
  • Abnormal bone marrow
  • Amyloidosis
  • Multiple myeloma
  • HIV/AIDS (3, 6)

Interpreting Results

Two test tubes; the one on the left remains blue which is negative and right is violet which is positive

Image 4: Two test tubes were subjected to biuret test. The other is heated, which changes the color of the mixture into violet. The unheated mixture remains blue in color.

Picture Source: slidesharecdn.com

  • No changes in color – If the color of the solution didn’t change, it is an indicator that there is no protein in a given sample.
  • Blue to violet/deep purple – If the solution changes from blue to violet or deep purple, it indicates that there is protein in the sample.
  • Blue to pink – If the solution changes in color from blue to pink, it indicates the presence of peptides or peptones. (1, 3, 6, 9, and 10)

References

  1. https://en.wikipedia.org/wiki/Biuret_test
  2. http://brilliantbiologystudent.weebly.com/biuret-test-for-protein.html
  3. https://www.onlinebiologynotes.com/biuret-test-principle-requirements-procedure-and-result/
  4. https://education.seattlepi.com/biuret-test-mean-biology-4659.html
  5. https://study.com/academy/lesson/what-does-biuret-test-for-method-equation.html
  6. https://onlinesciencenotes.com/biuret-test-principle-requirements-procedure-and-result-interpretation/
  7. http://dbpedia.org/page/Biuret_test
  8. https://www.ncbi.nlm.nih.gov/pubmed/11436195
  9. https://www.ruf.rice.edu/~bioslabs/methods/protein/biuret.html
  10. https://revisionworld.com/gcse-revision/applied-science/aqa-additional-applied-science/unit-2-exam-topics/food-science/food-tests/biuret-test-protein

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