Name: Amyrlicia B.

Date Performed: 11-29-23

Year and Course: 1st year & BSN Date Submitted: 12-03-23
Subject title: Biochemistry Code number: 2824
Name of Professor: Kent Charles Score:
Roque

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. To raise the pH of the solution to alkaline levels using KOH.

2. To form a coordination complex between the copper (II)
ions from CuSO4 and the nitrogen atoms involved in peptide
bonds.
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), 1 capsule glutathione, 1
capsule, collagen, gelatin solution (5 grams gelatin 100ml water)
 IV. PROCEDURE
Protein Sample Preparation
Albumin:
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.
Gelatin and Jelly powder:
1. Mix the gelatin and gulaman powder separately in a beaker and mix with 10-
20 ml of distilled water. You may add more water if the solution is still sticky.
2. Shake the contents thoroughly but try to avoid creating bubbles and leave it
for the next procedure.
Glutathione and collagen:
1. Mix the glutathione and collagen powder separately in a beaker and mix with
10-20 ml of distilled water. You may add more water if the solution is still
sticky.
2. Shake the contents thoroughly but try to avoid creating bubbles and leave it
for the next procedure.

The Biuret Test

1. Fill each test tube with one-third of the solutions to be tested (albumin, gelatin,
gulaman, glutathione and gelatin) . Prepare another tube 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 a 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

Sample Appearance Color Is protein
before adding Changes/Observation Present?
KOH and CuSO4
Albumin cloudy purple yes

Gelatin urine color purple yes

Gulaman cloudy white blue none
Glutathione GA: yellowish green/yellow none
G :transparent
Collagen C1:cloudy white purple yes
C2: cloudy
 1. During your preparation of albumin solution, what physical characteristics you
noticed in your albumin at room temperature?
We noted that the albumin solution was transparent at ambient
temperature. When we touched the solution, we observed that it was less
sticky than it had been before we added the distilled water. White
translucent-like particles were also floating about.

2. What is the proof or evidence in your experiment that your solution contains
proteins? Elaborate your answer.
When a drop of copper sulfate is added to an alkaline protein solution, it
produces a bluish-violet and intense purple hue. As a result of the creation
of violet hue, proteins are present.

3. What to you think are the reasons why your samples from protein sources
have remarkable differences in the results?
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4. Where do you think you can apply this activity in your future career? Or in
real-life situation?
We may apply this in our future careers as nurses by demonstrating and
training students on how to measure a substance's protein content.

VI. CONCLUSION

The protein testing experiment conducted by our group revealed that the
protein composition, presence of impurities, and handling of samples can
significantly impact the results obtained from various protein sources. We
observed remarkable differences in the behavior and reaction of different protein
sources due to their diverse types and amounts of proteins. The presence of
impurities or other substances in the protein samples also played a crucial role in
influencing the results. Moreover, the handling and preparation of the protein
samples could have also impacted the results. These variations can be attributed
to factors such as protein composition, the presence of impurities, and the
handling of the samples. Overall, this experiment has provided us with valuable
insights into the complexities of protein testing.