EXPERIMENT NO : 7

TITLE OF EXPERIMENT : DETERMINATION OF PROTEIN

NAME : NURIQMAL HAFIDZ BIN ZAMBRI

ID NUMBER : 2019434184

LECTURER’S NAME : MADAM DR. SHARIFAH RAINA

EXPERIMENT DATE : 28 NOVEMBER 2019

SUBMISSION DATE : 5 DECEMER 2019

INTRODUCTION

To determine the presence of protein in food sample we must conduct the

experiment name biuret test. The Biuret test for proteins may also be extended to
quantitatively measure the concentration of total protein using spectrometric methods.The
biuret test, also known as Piotrowski's test, is a chemical test used for detecting the
presence of peptide bonds. In the presence of peptides, a copper(II) ion forms mauve-
colored coordination complexes in an alkaline solution and food. Several variants on the test
have been developed, such as the BCA test and the Modified Lowry test.

The biuret reaction can be used to assess the concentration of proteins because
peptide bonds occur with the same frequency per amino acid in the peptide. The intensity of
the color, and hence the absorption at 540 nm, is directly proportional to the protein
concentration

Despite its name, the reagent does not in fact contain biuret ((H2N-CO-)2NH). The
test is named so because it also gives a positive reaction to the peptide-like bonds in the
biuret molecule.

In this assay, the copper(II) binds with nitrogens present in the peptides of proteins.
In a secondary reaction, the copper(II) is reduced to copper(I). Buffers, such as Tris and
ammonia interfere with this assay, therefore rendering this assay inappropriate for protein
samples purified from ammonium sulfate precipitation. Due to its insensitivity and little
interference by free amino acids, this assay is most useful for whole tissue samples and
other sources with high protein concentration.

An aqueous sample is treated with an equal volume of 1% strong base (sodium

hydroxide) followed by a few drops of aqueous copper(II) sulfate. If the solution turns purple,
it contains protein. 5–160 mg/mL can be determined. Peptides with the correct length of at
least 3 amino acids are necessary for a significant, measurable colour shift with these
reagents.
MATERIALS

a. Test tube
b. Test tube rack
c. Test tube holder
d. Measuring cylinder 10 ml
e. 10% NaOH solution
f. 1% CuSO4
g. Distilled water
h. Honey solution
i. Corn oil
j. Egg white solution
k. Dropper
l. Masking paper tape
m. Marker pen

PROCEDURE

1. The briefing on the lab will be given by the instructor.

2. Place four test tube in a test tube rack and label with Tube 1, Tube 2, Tube 3 and
Tube 4
3. Fill each tube with 2 mL of each solution:
a. Distilled water (Tube 1)
b. Honey solution (Tube 2)
c. Corn oil (Tube 3)
d. Egg white solution (Tube 4)
4. Add 2 mL of 10% NaOH solution to each test tube. WARNING! NaOH can cause
skin burns.
5. Add five drops of copper sulfate (1% CuSO 4) solution to each test tube and swirl to
mix the solution. Record the colour changes in all test tube.
6. Once the experiment is done, discard the content of the test tube and rinse them
properly and dry the test tube.
RESULT: Below are the table and picture of biuret test for determination of protein

Observation Interpretation

No Colour Change ( Blue ) Proteins are not present

No Colour Change (Yellow) Proteins are not present

Have two layer colour

First layer: (White) Proteins are not present
Second layer: (Blue)

Purple Proteins are present

Table 1: The observation and interpretation of biuret test for determination of protein
Figure 1: shown the test tube before add anything
Figure 2: shown the test tube after add 2 mL of 10% NaOH solution to each test tube
Figure 3: shown the test tube after add five drops of copper sulfate (1% CuSO 4) solution to
each test tube and swirl to mix the solution
DISCUSSION

Hydrated Copper sulphate. This provides the Cu (II) ions which form the chelate
complex. Cu (II) ions give the reagent its characteristic blue color.Potassium hydroxide
solution does not participate in the reaction but provides the alkaline medium.Potassium
sodium tartrate (KNaC4H4O6·4H2O) – stabilizes the chelate complex.

The Biuret test is based on the ability of Cu (II) ions to form a violet-coloured chelate
complex with peptide bonds (-CONH- groups) in alkaline conditions.Lone electron pairs
from 4 nitrogen atoms in the peptide bond coordinate a copper (II) ion to form the chelate
complex.

The chelate complex absorbs light at 540 nm so appears violet. Hence a color
change from blue to violet indicates that proteins are present.The greater the concentration
of peptide bonds, the greater the color intensity. If the concentration of peptide bonds is low
– such as when short-chain peptides are present - the color change is from blue to purple.

As 2 peptide bonds are required for the formation of the chelate complex, single
amino acids - no peptide bonds present - and dipeptides - only 1 peptide bond present –
give a negative result. According to the Beer-Lambert Law, the absorption of the sample is
directly proportional to the concentration of the species – in this case peptide bonds. Hence
absorption spectroscopy using spectrophotometer is a quantitative method which can be
used to determine the concentration of total protein, following the Biuret test.
CONCLUSION

This lab was a way to allow students to understand the structural and functional
roles ofmacromolecules. The lab results weren’t what was expected but it turned out
alright.The Biuret Test was the most difficult because of how little the food sample
changedcolor from the solution added. When the food sample didn’t change that meant
thatthere was an absence of that substance in the sample.The hypothesis was not
supported by the results. The hypothesis was that there would be traces of lipids, proteins,
and complex sugars in all of the food samples. There were traces of each in very few
samples from each test. What could have been done better for this test would have been
that there was barely enough time to do each experiment, maybe next time both groups
could split the work or there could be different tests other than the ones we used.
Task

 Indicate the colour changes in each tube

Test tube Colour present

Test tube 1 No Colour Change ( Blue )

Test tube 2 No Colour Change (Yellow)

Have two layer colour

Test tube 3 First layer: (White)
Second layer: (Blue)

Test tube 4 Purple

 Determine which food sample have the presence of protein.

White egg

 Explain how NaOH and CuSO4 react with protein and transformed the colour.
The Biuret reaction happens when Cu2+ - typically placed into solution as
cupric sulfate, is reduced to Cu1+ as it complexes with peptide bonds in proteins.
When it does so, the solution turns a purple color - lighter if there is less protein and
darker if there is more. Thus, a positive Biuret test will have turned a purple color.
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