AACB 3223 STRUCTURAL BIOCHEMISTRY

FACULTY OF APPLIED SCIENCES AND COMPUTING

KUALA LUMPUR CAMPUS

DIPLOMA IN SCIENCE
(CHEMISTRY AND BIOLOGY)
AACB 3223 STRUCTURAL BIOCHEMISTRY
LABORATORY MANUAL

Name
ID:
Lecturer:

Wong Chee Wein

13WAD01004 (AA1)
Dr. Loh Khye Er

AACB 3223 STRUCTURAL BIOCHEMISTRY

Title:

Test for Proteins

Objectives:
1. To detect the presence of peptide bond in protein by Biuret Test
2. To determine the protein with Millons Test
3. To identify the effect of heavy metals salt on protein

Introduction:
Proteins are flexible and dynamic macromolecules that come in a variety of shapes and sizes, and have a
very important role in biological processes. Proteins have a function in almost all processes within cells
and the body such as cell-signaling, metabolism, the cell cycle and many more.

Protein structure (general)

Proteins have four levels of structure
The first level of the protein structure is the primary structure which is the amino acid sequence.
The sequence of amino acids is based upon the codon sequence, a codon being a unit of three
nucleotides in the DNA or RNA sequence. In cells there are twenty common amino acids that are
used in the synthesis of proteins. Each amino acid also has a unique side chain giving it specific
properties; in addition they have an amino (N-terminus) and carboxylate (C-terminus) group
which form peptide bonds with other amino acids to form linear polypeptide chains that can
include over a hundred amino acids. A protein can consist of a single polypeptide or can be more
complex with several different polypeptides.
The secondary structure refers to interactions between amino acids in the polypeptide chain form
conformations such as alpha helices, beta strands and beta sheets (pleated sheet). These
conformations are stabilized by non-covalent hydrogen bonds between amide hydrogens and
oxygen in the amino acid backbone
The tertiary structure occurs when the secondary structure elements fold and compact. This
structure is stabilized via the non-covalent interactions of amino acids with its surrounding
environment, amino acids that are far apart in the primary structure can be brought into close
proximity.
Some proteins have a quaternary structure, which results through the interactions of two or more
of the same polypeptide or different polypeptide chain coming together to form a multi-subunit
protein..

AACB 3223 STRUCTURAL BIOCHEMISTRY

Peptide bonds
A peptide is a covalent bond that is formed between two molecules when the carboxyl group of one
molecule reacts with the amino group of another molecule, releasing a molecule of water. This is a
condensation reaction and usually occurs between amino acids. The resulting CO-NH bond is called a
peptide bond, and the resulting molecule is an amide.

Any number of amino acids can be joined together in chains of 50 amino acids called peptides,
50-100 amino acids called polypeptides, and over 100 amino acids called proteins. A number of
hormones, antibiotics, antitumor agents and neurotransmitters are peptides (proteins).
A peptide bond can be broken down by hydrolysis (the adding of water). The peptide bonds that
are formed within proteins have a tendency to break spontaneously when subjected to the
presence of water

Biuret Test
The Biuret reaction can be used for both qualitative and quantitative analysis of protein. The
biuret method depends on the presence of peptides bonds in proteins. When a solution of proteins
is treated with copper ions (Cu2+) in a moderately alkaline medium, a purple colored Cu2+peptide complex is formed which can be measured quantitatively by spectrophotometer in the
visible region. So, biuret reagent is alkaline copper sulfate solution.
The intensity of the color produced is proportional to the number of peptide bonds that are
reacting, and therefore to the number of protein molecules present in the reaction system. The
reaction do not occur with amino acids because the absence of peptide bonds, and also that with
di-peptide because presence of only one peptide bond, but do with tri-, oligo-, and poly-peptides.
Biuret reaction needs presence of at least two peptide bonds in a molecule .The reaction occurs
with any compound containing at least two bonds of

AACB 3223 STRUCTURAL BIOCHEMISTRY

Material & Methods:
I.

Biuret Test

3ml of soya bean extract is added into 4 different test tubes

3ml of 20% KOH is added in the first test tube and shake thoroughly

1ml of 0.5% of CuSO4 is added slowly drop by drop into the same
test tube

The development of color is noted and the observation is recorded

In tabular form

The steps are repeated with the substitution of distilled water and
1% of egg albumin. Any observations are recorded

AACB 3223 STRUCTURAL BIOCHEMISTRY

II.

Millons Test

2ml of soya bean extract is added into a test tube

A few drops of Millons reagent is added into the test tube and then
the sample is boiled in a water bath

The color change is noted and the observation is recorded in a

tabular form

The procedures are repeated with the substitution of distilled

water and 1% of egg albumin

AACB 3223 STRUCTURAL BIOCHEMISTRY

III.

Protein precipitation by Heavy Metals

3 ml of protein extract is pipetted into 4 separated test tubes

The 1st test tube is added with 2 drops of 1% HgCl2

The result is then observed and recorded in a table before adding

1% of HgCl2 in excess

The formation of precipitation is noted and the amount of

precipitation on a relative basis (none, very slightly, slightly, etc) is
recorded.

The procedure is then repeated for 3 remaining tubes using 1% of

AgNO3, 1% of MgCl2 , and 1% of CuSO4

The steps are repeated with 2 % of egg albumin in place of the

protein extract.

AACB 3223 STRUCTURAL BIOCHEMISTRY

Result:
B. Protein Test (Biuret Test)
Test tubes
1st

Solutions
Soya bean extract

Observations
Milky brown suspension
is formed with
foam on top of
solution

2nd

Distilled Water

Very fade crystal

blue color solution
is formed
( negative result)

3rd

1% Egg Albumin

Foam is formed
on top of solution
purple violet can be
seen clearly
( positive result)

Figure 1: From left (soya bean extract), second from left, distilled water, and 1% of egg
albumin

AACB 3223 STRUCTURAL BIOCHEMISTRY

Millons Test
Test tubes

Observation

1st

Solutions
1% of egg
albumin

2nd

Distilled water

crystal clean colorless solution

with no precipitate

3rd

Soya bean extract

2 layers of dry precipitate is

are formed, pinkish aggregate
at the bottom while milky white
aggregate on top of the mixture

Pink color aggregation is formed

on top of solution clumping
Together
( positive result)

AACB 3223 STRUCTURAL BIOCHEMISTRY

Protein Precipitation by Heavy Metals
Protein Extracts
Number of drops
Test
Tubes
1st

2nd

3rd

4th

Reagent
Solutions
HgCl2

CuSO4

AgNO3

MgCl2

2 Drops
Non suspension
Milky White
solution
is formed
Non suspension
Milky white
solution
is formed
No precipitate is
formed, a very
slight
suspension
No precipitate is
formed, a very
slight
suspension

Added in Excess
Foamy is seen on top of solution
2 layers, very slightly cloudy on top
white precipitate is formed
2 Layers precipitate is formed
foamy, greenish color precipitate
2 layers of precipitate is formed,
thick purplish powdery precipitate
present
Foamy on top of solution, no visible
layers of precipitation, cloudy white
solution

Figure 2: From left HgCl2, second from left, AgNO3, follow by MgCl2, and CuSO4

AACB 3223 STRUCTURAL BIOCHEMISTRY

2% Egg Albumin

Test
Tubes
1st

Solutions
HgCl2

2nd

CuSO4

3rd

AgNO3

4th

MgCl2

Number of drops
2
Added
in
Drops
Excess
Non precipitate
2 layers of precipitate, slightly
white milky precipitate is
White cloudy
formed
suspension
Milky white cloudy
Crystal clear solution with no
precipitate, solution is slightly
suspension
crystal
blue color
2 layers of precipitate, slightly
Non precipitate
white
milky precipitate Is
White cloudy
present
suspension
Foamy on top of solution,
Colorless solution
colorless
solution

Figure 2: From left HgCl2, second from left, AgNO3, follow by MgCl2,
and CuSO4

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AACB 3223 STRUCTURAL BIOCHEMISTRY

Discussion:
Millons Test
Biuret Test served as a purpose of determining the properties of protein same goes to
Millons Test, in Millons Test, it is considered as a general protein test and not a specific
test for protein, it functions as a phenolic compound detector whereby it shows positive
result when phenolic compound is present in the structure and when it is soluble, it gives a
dark red or pinkish solution.

Figure shows the structure of phenolic compound presence in protein

Biuret Test
Biuret Test, however is a much more specific test for protein, as it serves as a detector for
peptide bond, when tested positive, it will change from blue to purple violet. In this
reaction, proteins form a purple colored complex with CuSO4 in a strongly alkaline solution.
When proteins and peptides (peptide bonds) treated with an alkaline solution of dilute
copper sulfate a violet color is formed. A positive test is indicated by the formation of a
violet color.

Figure shows the peptide bond formed between 2 amino acids by removal of water

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AACB 3223 STRUCTURAL BIOCHEMISTRY

Protein Precipitation by Heavy Metals
Since salts are ionic they disrupt salt bridges in proteins. The reaction of a heavy metal salt
with a protein usually leads to an insoluble metal protein salt. Heavy metal salt will
neutralize the protein. By the negative charge of protein will bind with positive charge of
metal ion. Then the protein will precipitate as insoluble metal protein salt.

Conclusion:
In this experiment, the test for 1% egg albumin by using Biuret Test shows the positive
result of purple violet solution this shows that, peptide bonds is presence in 1 % egg
albumin thats why the solutions turned purple violet color. As for Millons Test, the
experiment is also a success where it shows the presence of pinkish red aggregate on top of
the solution,

References:
1. http://biochemistrygirls.blogspot.com/2013/04/experiment-2-proteinexperiment.html
2. http://shinaebiochem.blogspot.com/2013/04/practical-2-proteinexperiment.html#
3. http://www.fastbleep.com/biology-notes/40/116/780
4. http://www.peptideguide.com/peptide-bond.html
5. http://fac.ksu.edu.sa/sites/default/files/Qualitative_chemical_reaction_of_fun
ctional_group_in_protein.pdf

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