Teacher’s Guide: Detecting Tyrosine in Proteins Using Millon's Reagent and Protein

Detection with Biuret Reagent

Aim
1. To detect the presence of tyrosine in various household protein samples using Millon's
reagent without the need for heating.
2. To confirm the presence of proteins in these samples using Biuret reagent.
Materials
 Millon's reagent
 Biuret reagent
 Distilled water
 Test tubes or small glass containers
 Dropper or pipette
 Household protein samples:
 Milk
 Egg white
 Soy milk
 Cheese
 Yogurt
 Labels and marker
Procedure
Part 1: Detecting Tyrosine with Millon's Reagent
1. Prepare the Samples:
 Milk: Use 1-2 mL directly.
 Egg White: Dilute 1-2 mL of egg white with an equal volume of distilled
water.
 Soy Milk: Use 1-2 mL directly.
 Cheese: Grate a small piece of cheese and dissolve in 5 mL of distilled water.
Use 1-2 mL of the solution.
 Yogurt: Dilute 1-2 mL of yogurt with an equal volume of distilled water.
2. Set Up the Test:
 Label each test tube with the name of the sample.
  Add 1-2 mL of each prepared sample to its respective test tube.
3. Adding Millon's Reagent:
 Add 1-2 mL of Millon's reagent to each test tube containing the sample.
 Mix gently by swirling the test tube.
4. Observing Results:
 Let the reaction sit for about 10-15 minutes at room temperature.
 Observe any color changes. A positive reaction will usually result in a reddish-
brown color.
Part 2: Confirming Protein Presence with Biuret Reagent
1. Prepare the Samples:
 Use the same prepared samples from Part 1.
2. Set Up the Test:
 Label a new set of test tubes with the name of the sample.
 Add 1-2 mL of each prepared sample to its respective test tube.
3. Adding Biuret Reagent:
 Add 1-2 mL of Biuret reagent to each test tube containing the sample.
 Mix gently by swirling the test tube.
4. Observing Results:
 Let the reaction sit for about 5 minutes at room temperature.
 Observe any color changes. A positive reaction for proteins will usually result
in a violet or purple color.
Predicted Results and Explanation
Millon's Reagent Test

Initial Color After Adding Tyrosine Presence

Sample Color Millon's Reagent (Yes/No) Explanation

Milk contains casein, a protein rich in

Milk White Reddish-Brown Yes tyrosine.

Egg Egg whites contain albumin, which has

White Clear Reddish-Brown Yes tyrosine residues.

Soy Milk Pale Yellow No significant change No Soy protein has lower tyrosine content
 Initial Color After Adding Tyrosine Presence
Sample Color Millon's Reagent (Yes/No) Explanation

compared to animal proteins.

Cheese Yellowish Reddish-Brown Yes Cheese, like milk, contains casein.

Yogurt contains casein and whey proteins,

Yogurt White Reddish-Brown Yes both of which have tyrosine.

Biuret Reagent Test

Initial Color After Adding Protein Presence

Sample Color Biuret Reagent (Yes/No) Explanation

Milk proteins react positively with Biuret

Milk White Violet Yes reagent.

Egg Egg white proteins react positively,

White Clear Violet Yes confirming their presence.

Despite low tyrosine, soy protein still

Soy Milk Pale Yellow Violet Yes shows a positive Biuret reaction.

Cheese proteins also react positively with

Cheese Yellowish Violet Yes Biuret reagent.

Yogurt proteins confirm the presence of

Yogurt White Violet Yes protein.

Discussion Questions and Answers

1. Which samples showed a positive reaction for tyrosine with Millon's reagent?
Explain the significance of these results.
Answer:
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Milk, egg white, cheese, and yogurt showed a positive reaction for tyrosine. These results are
significant because they indicate that these samples contain proteins with tyrosine residues.
The presence of tyrosine is confirmed by the reddish-brown color change upon reaction with
Millon's reagent.
2. How does the dilution of the protein samples affect the intensity of the color
change in both tests?
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The dilution of protein samples generally reduces the intensity of the color change in both
tests. A more concentrated protein sample would produce a more intense color change due to
a higher number of reactive sites (tyrosine in Millon's test, peptide bonds in Biuret test) being
available for the reaction.
3. Compare the results from the Millon's reagent test and the Biuret reagent test.
What conclusions can you draw about the protein content and the presence of
tyrosine in these samples?
Answer:
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The Biuret reagent test confirms the presence of proteins in all samples (milk, egg whit
Further Questions to Ask in Class
1. Why might soy milk show a lower intensity of color change with Millon's reagent
compared to milk and cheese?
 Answer: Soy milk contains proteins with lower tyrosine content compared to
animal-based proteins found in milk and cheese. Tyrosine is the amino acid
that reacts with Millon's reagent to produce the reddish-brown color. The
difference in tyrosine content accounts for the variation in color intensity
observed.
2. How could you modify the experiment to quantify the amount of tyrosine in each
sample?
 Answer: To quantify the amount of tyrosine, students could perform a
spectrophotometric analysis. This involves measuring the absorbance of the
sample at a specific wavelength corresponding to the color of the tyrosine-
Millon's reagent complex. By comparing the absorbance values to a standard
curve prepared from known concentrations of tyrosine, the amount of tyrosine
in each sample can be determined.
3. What other amino acids could be detected using specific reagents, and what
would those tests look like?
 Answer: Various amino acids can be detected using specific reagents. For
example:
 Ninhydrin reagent can be used to detect amino acids with primary
amino groups, such as lysine and arginine, by forming colored
complexes.
  Sakaguchi reagent can detect ornithine, a non-protein amino acid, by
forming a purple-colored complex.
 Xanthoproteic test can detect amino acids with aromatic rings, such as
phenylalanine and tryptophan, by forming yellow-colored compounds
upon nitration followed by the addition of alkali.
4. How does the presence of proteins in these household items relate to their
nutritional value?
 Answer: Proteins are essential macronutrients that play vital roles in various
physiological processes, including tissue repair, enzyme function, and immune
system function. The presence of proteins in household items such as milk,
cheese, and yogurt contributes to their nutritional value by providing essential
amino acids necessary for maintaining overall health and well-being.
5. What are some real-world applications of these types of biochemical tests?
 Answer: Biochemical tests like the ones performed in this experiment have
numerous real-world applications:
 Food industry: Testing for protein content in food products to ensure
quality and nutritional labeling accuracy.
 Clinical diagnostics: Detecting specific proteins or amino acids in
bodily fluids for disease diagnosis and monitoring.
 Forensic analysis: Identifying protein residues at crime scenes for
evidence collection and analysis.
 Environmental monitoring: Assessing protein levels in environmental
samples to evaluate ecosystem health and pollution levels.