Name: _____________________________
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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:
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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.
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4.
The Biuret Test
Fill a test tube one-third of the solution to be tested (albumin solution). Prepare
another tubes as your control with distilled water only.
Add 10 drops of KOH to each tube and shake them thoroughly.
Add copper sulfate solution one drop at the time. Shake the solution after each
drop. This is very important to avoid coagulation.
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?
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2. What is the proof or evidence in your experiment that your solution contains
proteins? Elaborate your answer.
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3. Where do you think you can apply this activity in your future career? Or in reallife situation?
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VI. CONCLUSION
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LIBRARY WORK (Kindly submit this portion individually)
 In an A4 bond paper research at least two (2) journals / articles about proteins
(Times New Roman font 13, landscape)
Author(s)
Year
published
Title
Methods
Results
Note: In the submission of your answers in LMS, two documents must be seen, the
lab activity and the library work.