Exercise no.

6
BIURET TEST FOR THE PRESENCE OF PROTEINS

I. INTRODUCTION

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
coordinate with the nitrogen atoms of these peptide bonds, the color of the solution
change 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.

II. OBJECTIVES:

At the end of the experiment, each student will be able to:

1. Test the presence of proteins with the use of biuret.

2. Know the procedure and perform the protein sample preparation.

III. MATERIALS:
1-200ml beaker, stirring rod, 3-test tubes, test tube rack, test tube holder, test
tube brush, 2 medicine dropper, 50ml distilled water, 2ml KOH solution, 2ml
CuSO4 solution, 2- egg (raw)

IV. PROCEDURE
Protein Sample Preparation
 1. Separate the egg albumin and the egg yolk: In this experiment, you will use only
the albumin (egg white).
2. Place the albumin in the beaker and mix with 10-20 ml of distilled water. You
may add more water if the solution is still sticky.
3. Shake the contents thoroughly but try to avoid creating bubbles and leave it for
the next procedure.

The Biuret Test

1. Fill a test tube one-third of the solution to be tested (albumin solution). Prepare
another tubes as your control with distilled water only.
2. Add 10 drops of KOH to each tube and shake them thoroughly.
3. Add copper sulfate solution one drop at the time. Shake the solution after each
drop. This is very important to avoid coagulation.
4. Keep adding the copper sulfate solution until you will notice color change,
usually after 5-9 drops

Note: solution containing a long-chain protein will turn a vivid purple when exposed to the Biuret test. A
solution containing a shorter protein chains will turn into pink. A solution having no protein will turn
cloudy blue

V. RESULTS AND OBSERVATION

1. During your preparation of albumin solution, what physical characteristics you

noticed in your albumin at room temperature?

When cooked, heat promotes denaturation, which hardens the albumen and
turns it opaque white. The denaturation of the egg albumin protein is reflected in the
changes in viscosity and color. Albumins are a type of water-soluble protein that can be
found in egg white, milk, and blood serum. Albuminoids are any compounds that
contain albumins. Although an egg white is mostly water, proteins such as albumins,
globulins, and mucoproteins make up around 10% of the clear and viscous substance.
Because of its nutritional benefits and capacity to thermally coagulate and bind
substances together, egg albumin protein is a beneficial ingredient in many food items,
including pet food and pet treats.

2. What is the proof or evidence in your experiment that your solution contains
proteins? Elaborate your answer.

What is the proof or evidence in your experiment that your solution contains
proteins? Elaborate your answer. Egg white is used as a sample to determine the
presence of protein. It was placed in a test tube. After that, dilute sodium hydroxide was
added and also copper sulfate was added in a drop by drop shaking the test tube every
after one drop. As the copper sulfate was being added in the solution, we can notice
that there is a color change. A violet coloration appears which indicates the presence of
proteins. The sample becoming violet in color is the evidence that it has proteins.
 3. Where do you think you can apply this activity in your future career? Or in real-
life situation?

As a pharmacist in the future, there is a big possibility that we can be part of a

team that would produce drugs. Vaccines are very important because they are the ones
that would stop or prevent the spreading of any diseases that are dangerous and
contagious. That being said, proteins are used as an antibody to develop vaccines. This is
practically needed especially in the pandemic that we are going through right now. The
application of the protein is also needed for the nutritionist or others that are
manufacturing foods if they want to make sure that the food, they are making has a
protein.

VI. CONCLUSION

A Biuret test is a chemical test that detects the existence of peptide bonds in a
material. The color intensity is related to the number of peptide bonds present in the
protein molecule that is reacting as well as the number of protein molecules present in the
reaction system. The biuret test is a chemical assay that identifies protein content in a
sample. The presence of proteins is confirmed by a color change in the test. The sample
will become violet if proteins are detected.