Qualitative protein analysis using nitric acid

By Jillian Jastrzembski

The xanthoproteic test is a qualitative analytical assay to determine the presence of proteins and amino acids in a sample. This simple test uses nitric acid along with a strong base to induce a colorimetric change in the presence of certain amino acids.

In this article, we’ll talk about why this test is important, how it works, and how to perform it.

Why do we need qualitative protein analysis?

It is difficult to overstate the importance of proteins to biology. In fact, the word protein itself comes from the Greek word proteios, meaning “of primary importance.”

Proteins are responsible for all of the physiological processes and chemical reactions that are essential to life. They make up cell structure and muscle tissue, regulate gene expression, and regulate the immune system. Proteins are comprised of amino acids, which also make up hormones and neurotransmitters.

There are 20 amino acids, which are linked together via peptide bonds to form proteins. Each amino acid has a unique structure, and contributes to a unique function in the body. Being able to test for the presence of amino acids – and to differentiate between them – is therefore extremely important.

Proteins and amino acids in food and other agricultural products have a huge impact on nutritional value, as well as the physiochemical properties of food. Protein assays are also useful for clinical and diagnostic purposes. Just as protein is critical to life, the presence of a protein mistake or malfunction can indicate disease or underlying health condition.

What is the xanthoproteic test?

The xanthoproteic test is a simple, qualitative assay that can detect the presence of amino acids containing an aromatic ring or benzene group. This includes amino acids such as tyrosine, tryptophan, and phenylalanine.

The test requires the use of concentrated nitric acid, which is reacted with the sample in the presence of heat. Under these conditions, the aromatic amino acids will form a white or yellow deposit called a “xanthoprotein.” (The prefix xantho means yellow.)

The color change is a result of the nitration reaction. That means that the nitrate from nitric acid binds on the aromatic ring of the amino acid. The resulting molecule has a yellowish hue that was absent in the starting product.

The next step is to add a strong base, which could be sodium hydroxide or ammonia. This creates a more dramatic colorimetric change, as the xanthoprotein takes on a deep orange hue. However, this step is not strictly necessary.

It is possible to differentiate between tyrosine and tryptophan based on the exact color change, which can be matched to a known analytical standard. However, phenylalanine gives a weak response if any, despite the presence of an aromatic ring.

Advantages of this test are that it is semi-specific, because it tests only for amino acids which contain an aromatic ring. It is also relatively simple and quick to perform, and does not require special equipment. Because it is only a qualitative test, it does not allow for quantitation of the amino acids present. In other words, you will be able to test whether or not they are there, but you cannot determine how much is there. The xanthoproteic test can also be combined with other simple protein assays to increase specificity.

How to perform the xanthoprotein test

This is also an appropriate experiment for a classroom setting. You can perform the assay on egg whites to test for the presence of tryptophan and tyrosine.

You will need:

• Egg whites (albumin) or other protein solution
• Concentrated nitric acid
• Concentrated sodium hydroxide or ammonia

Add 2 drops of concentrated nitric acid to the protein solution. Heat the solution in a water bath. The solution should change color if the test is positive. Cool the solution before adding a few drops of the basic solution (optional). The yellow color will deepen or turn orange.

Summary

Nitric acid reacts with certain amino acids to form a compound with a yellowish hue. The xanthoproteic test is a simple, qualitative assay that takes advantage of this reaction and color change. By adding concentrated nitric acid to a sample in the presence of heat and observing for a color change, one can test for the presence of amino acids that contain a benzene ring, including tryptophan and tyrosine.

References

Asthana, S. K. M. A., Maurya, S. P., Maurya, P., & Maurya, A. (2019). Qualitatve analysis of protein: egg albumin and milk. Indian Journal of Drugs, 7(1), 30-33.

Kamineni, S., Manepally, M., & Kamineni, E. P. (2016). Musculoskeletal protein analysis techniques—a review. J Rheumatol Arthritic Dis, 1(1), 1-9.

Poos, M. I., Costello, R., & Carlson-Newberry, S. J. (1999). Conclusions and Recommendations from the Workshop Report The Role of Protein and Amino Acids in Sustaining and Enhancing Performance Submitted June 1999. In Committee on Military Nutrition Research: Activity Report: December 1, 1994 through May 31, 1999. National Academies Press (US).

Subroto, E., Lembong, E., Filianty, F., Indiarto, R., Primalia, G., Putri, M. S. K. Z., … & Junar, S. (2020). The analysis techniques of amino acid and protein in food and agricultural products. Int. J. Sci. Technol. Res, 9(10), 29-36.

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