Experiment Report

Determination the Content of

Protein by Using Biuret Method

Submitted by:
Tarisa Adani

(12030194231)

INTERNATIONAL CHEMISTRY EDUCATION 2012

MATHEMATCS AND NATURAL SCIENCE FACULTY
DEPARTMENT OF CHEMISTRY
2013

A. Date of Experiment : November, 2014

B. Title of Experiment : Determination the content of protein by using
biuret method
C. Goal of Experiment : To determine the content of protein in sample
(duck egg) by using biuret method
D. Basic Theory

Protein
Protein is found throughout the bodyin muscle,
bone, skin, hair, and virtually every other body part or
tissue. It makes up the enzymes that power many chemical
reactions and the hemoglobin that carries oxygen in your
blood. At least 10,000 different proteins make you what
you are and keep you that way.
Protein is built from building blocks called amino acids. Our bodies
make amino acids in two different ways: Either from scratch, or by
modifying others. A few amino acids (known as the essential amino acids)
must come from food.

Animal sources of protein tend to deliver all the amino acids we need.

Other protein sources, such as fruits, vegetables, grains, nuts and

seeds, lack one or more essential amino acids.
Proteins are polymers of amino acids covalently linked through

peptide bonds into a chain. Within and outside of cells, proteins serve a
myriad of functions, including structural roles (cytoskeleton), as catalysts
(enzymes), transporter to ferry ions and molecules across membranes, and
hormones to name just a few.
With few exceptions, biotechnology is about understanding,
modifying and ultimately exploiting proteins for new and useful purposes.
To accomplish these goals, one would like to have a firm grasp of protein
structure and how structure relates to function. This goal is, of course,

much easier to articulate than to realize! The objective of this brief review
is to summarize only the fundamental concepts of protein structure.
Amino Acids
Proteins are polymers of amino acids joined
together by peptide bonds. There are 20 different amino
acids that make up essentially all proteins on earth.
Each of these amino acids has a fundamental design
composed of a central carbon (also called the alpha carbon) bonded to:

a hydrogen

a carboxyl group

an amino group

a unique side chain or R-group

Thus, the characteristic that distinguishes one amino acid from

another is its unique side chain, and it is the side chain that dictates an
amino acids chemical properties. Examples of three amino acids are shown
below, and structures of all 20 are available. Note that the amino acids are
shown with the amino and carboxyl groups ionized, as they are at
physiologic pH.

Except for glycine, which has a hydrogen as its R-group, there is

asymmetry about the alpha carbon in all amino acids. Because of this, all
amino acids except glycine can exist in either of two mirror-image forms.
The two forms - called stereoisomers - are referred to as D and L amino

acids. With rare exceptions, all of the amino acids in proteins are L amino
acids.
The unique side chains confer unique chemical properties on amino
acids, and dictate how each amino acid interacts with the others in a
protein. Amino acids can thus be classified as being hydrophobic versus
hydrophilic, and uncharged versus positively-charged versus negativelycharged. Ultimately, the three dimensional conformation of a protein - and
its activity - is determined by complex interactions among side chains.
Some aspects of protein structure can be deduced by examining the
properties of clusters of amino acids. For example, a computer program
that plots the hydrophobicity profile is often used to predict membranespanning regions of a protein or regions that are likely to be immunogenic.

Levels of Protein Structure

Structural features of proteins are usually described at four levels of
complexity:

Primary structure: the linear arrangment of amino acids in a protein

and the location of covalent linkages such as disulfide bonds between
amino acids.

Secondary structure: areas of folding or coiling within a protein;

examples include alpha helices and pleated sheets, which are
stabilized by hydrogen bonding.

Tertiary structure: the final three-dimensional structure of a protein,

which results from a large number of non-covalent interactions
between amino acids.

Quaternary structure: non-covalent interactions that bind multiple

polypeptides into a single, larger protein. Hemoglobin has quaternary
structure due to association of two alpha globin and two beta globin
polyproteins.

The primary structure of a protein can readily be deduced from the

nucleotide sequence of the corresponding messenger RNA. Based on
primary structure, many features of secondary structure can be predicted
with the aid of computer programs. However, predicting protein tertiary
structure remains a very tough problem, although some progress has been
made in this important area.
Duck Egg
Duck eggs boost your vitamin intake and provide
considerable amounts of vitamins A and B-12. The vitamin
A from your diet promotes new cell development to keep
your tissues healthy and also maintains good eyesight. A
duck egg contains 472 international units of vitamin A -one-fifth of the recommended daily intake for women and
16 percent for men. The vitamin B-12 in duck eggs keeps
your nerves healthy and promotes red blood cell function.
Each duck egg boasts 3.8 micrograms of vitamin B-12,
more than your entire daily recommended B-12 intake. It
also contains small amounts of several B-complex vitamins,
as well as vitamins D and E.
Duck eggs also offer nutritional value because of
their selenium and iron content. Selenium supports healthy
immune function and helps you make thyroid hormones.
Iron helps your red blood cells carry oxygen and plays a
role in energy production. Each duck egg contains 2.7
milligrams of iron -- 34 percent of the recommended daily

intake for men and 15 percent for women -- as well as 25.5

micrograms of selenium, or 46 percent of your intake
requirement. Duck eggs also contain small amounts of zinc,
phosphorus and calcium.
Duck eggs' high cholesterol content means you
should consume them in moderation. Each egg contains
619 milligrams of cholesterol, which is more than twice the
daily recommended limit, or more than three times the
limit for those suffering from high cholesterol or heart
disease. A high cholesterol intake can negatively affect
your blood cholesterol levels. Limit your cholesterol intake
when eating duck eggs by limiting your portion size to one
egg and pairing it with cholesterol-free ingredients, such as
vegetables

and

fruits.

If

you're

sensitive

to

dietary

cholesterol, you might want to avoid duck eggs entirely.

Biuret Method
The Biuret Method, which is the most widely used method for total
protein determination, relies on the complexation of Cu 2+ by the function
groups involved with the peptide bond. A minimum of two peptide bonds
is needed for the complexation to occur. Upon complexation, a violet color
is observed. The absorbance of the Cu2+-protein complex is measured at
520 nm and compared to a standard curve.
E. Tools and Materials :
-

Test tube

Standard protein solution

Spectronic UV-Vis

Biuret reagent

F. Procedure of Experiment

Preparation of Sample
Liquid Sample
-

Diluted sufficiently

Sample

Tube I

Preparation of Standard

Tube II

- Added 1 mL
standard solution
with the content
1 mg/mL protein

- Added 1 mL
standard solution
with the content
2 mg/mL protein

Tube III

Tube IV

- Added 1 mL
standard solution
with the content
3 mg/mL protein

- Added 1 mL
standard solution
with the content
4 mg/mL protein

Tube V
- Added 1 mL
standard solution
with the content
5 mg/mL protein

Added 4 mL biuret reagent into each

tube, mixed.
Incubated those test tube at 37oC for 10
minutes / at room temperature for 30
minutes.

Stable Purple Form

-

Result

Measured the absorbance of each

solution in test tube at wavelength 520
nm by using spectronic UV-Vis

Determintion the Absorbance of Blanko Solution

1 mL aquades
-

Entered to test tube

Added 4 mL biuret reagent into that tube and shake
Incubated that tube at temperature 37oC for 10 minutes
or at room temperature for 30 minutes
Measured the absorbance of the solution at that tube, at
wavelength 520 nm with spectronic UV-Vis

Result

Determination the Absorbance of Sample Solution

1 mL sample
-

Result

Entered to test tube

Added 4 mL biuret reagent into that tube and shake
Incubated that tube at temperature 37oC for 10 minutes
or at room temperature for 30 minutes
Measured the absorbance of the solution at that tube, at
wavelength 520 nm with spectronic UV-Vis

G. Table of Experiment
N
o.
1.

Procedur
Sample Preparation
Liquid Sample
(duck egg protein)
Diluted sufficiently
Sample

Experiment Result
Before:
Duck Egg protein :
colorless thick
Aquades : colorless
Biuret : blue
After :
Duck egg
protein+aquades:
colorless
Duck egg
protein+aquades+
filter: colorless
Duck egg
protein+aquades+
flter+ biuret : blue
solution

Hypothesis/Reaction
Sample protein color is
colorless

Conclusion
Sample protein
color is colorless.

2.

Making Standard
Tube I

Tube
II

- Added 1
mL
standard
solution
with the
content 1
mg/mL
protein

Tube
III

- Added
1 mL
standard
solution
with the
content 2
mg/mL
protein

Tube
IV

- Added 1
mL
standard
solution
with the
content 3
mg/mL
protein

Before :
Sample : colorless
Biuret : blue solution

Tube
V

- Added 1
mL
standard
solution
with the
content 4
mg/mL
protein

- Added 1
mL
standard
solution
with the
content 5
mg/mL
protein

Added 4 mL biuret reagent

into each tube, mixed.
Incubated those test tube at
37oC for 10 minutes / at room
temperature for 30 minutes.
Stable Purple Form
Measured the absorbance of
each solution in test tube at
wavelength 520 nm by using
spectronic UV-Vis
Result

After :
Sample+biuret :
1 mg : blue
2 mg : purplish blue
3 mg : violet
4 mg : violet (++)
5 mg : violet (+++)

Cu2+ base solution,

formed complex with
peptide bonding so
produce violet color from
absorbantion 520 nm
wavelength

Protein
concentration
proportional
with the
maximum
wavelength
The higher
concentration
the violet
(darken) of the
solution
The linear
equation from
the protein
standard curve
is
y=
0.044x+0.008
R2 =0.980

3.

Determination the Absorbantion of

Blanko Solution
1 mL aquades
Entered to test tube
Added 4 mL biuret reagent into that tube and shake
Incubated that tube at temperature 37oC for 10 minutes or at
room temperature for 30 minutes
Measured the absorbance of the solution at that tube, at
wavelength 520 nm with spectronic UV-Vis

Result

Before :
Sample : colorless
Biuret : blue solution
Aquades : colorless
After :
Aquades+Biuret
(blanko solution) :
blue solution

Blanco solution doesnt contain Blanco solution

protein
doesnt contain
protein

4.

Determining The Absorbantion of sample

solution
1 mL sample
Entered to test tube
Added 4 mL biuret reagent into that tube and shake
Incubated that tube at temperature 37oC for 10 minutes or at
room temperature for 30 minutes
Measured the absorbance of the solution at that tube, at
wavelength 520 nm with spectronic UV-Vis

Result

Before :
Sample : colorless
Biuret : blue solution
Aquades : colorless
After
Sample+aquades :
colorless
Sample+biuret :
blue solution
Sample+biuret+inc
ubation : blue
solution

The perfect dilution, the

perfect absorbantion
result.

The equation
based on the
curve is
y= 0.044x+0.008
The Biuret reagent is R2 =0.980
made
of
sodium
hydroxide (NaOH) and Sample
hydrated
copper(II) concentration :
sulfate, together with 1. 1.34
potassium
sodium 2. 1.59
[4]
tartrate.
Potassium 3. 1.20
sodium
tartrate[5]
is
added to complex to The average of
stabilize the cupric ions. sample
Proteins in the alkaline concentration is
environment reduce Cu2+ 1.38
to Cu+, which forms a
coordination
complex
with proteins, leading to
a blue to light violet
color change.

H. Analysis and Discussion

White egg is a general name for the colorless liquid in

an albumen or white egg substance (glaire). White egg is an
eggs citoplasm, which is until fertility is a cell (contain of
yellow egg). White egg is about 2/3 from the total weigh of the
egg and almost 90% from the weigh is water. And another
weigh is from protein, mineral, fat, vitamin, and glucose.
The content of protein is the amount of protein contain
in material which is described in percentage. This experiment
is about determination of the content of protein by using
biuret method.
This experiment started with made a sample from
duck egg. We just need the white egg and then we diluted it.
After we get that sample, we take 1 mL of sample to enter it
into test tube. Then, we add 4 mL of biuret reagent and mix it.
The result after we add the biuret reagent is the solution
becomes blue.
The Biuret reagent is made of sodium hydroxide
(NaOH)

and

hydrated

copper(II)

sulfate,

together

with

potassium sodium tartrate.[4] Potassium sodium tartrate[5] is

added to complex to stabilize the cupric ions. Proteins in the
alkaline environment reduce Cu2+ to Cu+, which forms a
coordination complex with proteins, leading to a blue to light
violet color change.
And then, we incubate the solution at temperature
37oC for 10 minutes. Then, we measure the absorbance of the
solution at that tube, at wavelength 520 nm with spectronic
UV-Vis. From there, we get the equation based on the curve is
y= 0.044x+0.008 and R2 =0.980. the concentration of
samples are for test tube 1 we get 1.34, test tube 2 is 1.59,
and test tube 3 is 1.20. Then we get the average of sample
concentration is 1.38.

For blanko solution, we fill 1 mL of aquades into test

tube and then we add 4 mL of biuret reagent. It produce a
blue solution. And then we incubated that tube at temperature
370C for 10 minutes and then measure the absorbance of the
solution at wavelength 520 nm with spectronic UV-Vis. From
the result that we get, we can conclude that blanko solution
doesnt contain of protein.
And the last is making standards. We take 5 test tube
and then fill it with 1 mL of standard protein solutions with the
content is 1 mg, 2 mg, 3 mg, 4 mg, 5 mg per mL protein. And
then we add 4 mL of biuret reagent into each tubes and mix it.
The result that we get are: when we mix the standard protein
solution with the content is 1 mg with biuret reagent is the
solution change into blue, when we mix the standard protein
solution with the content is 2 mg with biuret reagent is the
solution change into purplish blue, when we mix the standard
protein solution with the content is 3 mg with biuret reagent is
the solution change into violet, when we mix the standard
protein solution with the content is 4 mg with biuret reagent is
the solution change into violet (++), and when we mix the
standard protein solution with the content is 5 mg with biuret
reagent is the solution change into violet (+++). Cu2+ base
solution, formed complex with peptide bonding so produce
violet color from absorbantion 520 nm wavelength. The linear
equation from the protein standard curve is y= 0.044x+0.008
and R2 =0.980.

I. Conclusion
-

Protein concentration proportional with the maximum

wavelength

The higher concentration the violet (darken) of the

solution

The linear equation from the protein standard curve is

y= 0.044x+0.008
R2 =0.980

The equation based on the curve is

y= 0.044x+0.008
R2 =0.980

Sample concentration :
1. 1.34
2. 1.59
3. 1.20
The average of sample concentration is 1.38

J. Attachment

:
Biuret reagent

Diluted sample with aquades

Incubated

Sample A, B, C and blanko

after added biuret reagent

Sample A, B, C and blanko

after incubated

Standard solution before

incubated

Standard solution after

incubated

K. Task :
1. Make the standard curve of concentration vs absorbance.
By using that standard curve determine the content of
protein of sample!
2. Is peptida will give a positive reaction for biuret reagent? If
it is true, how to determine the content of protein mixed
with peptida?
Answer:
1.
concentrati Waveleng
on
0
1
2
3
4
5

th
0
0,049
0,108
0,154
0,196
0,215

Y = 0,044x + 0,008

Y = 0,044x + 0,008

0,067 = 0,044x + 0,008

0,078 = 0,044x +

0,008
0,059 = 0,044x

0,07 = 0,044x

X = 1,34

x = 1,59

Y = 0,044x + 0,008
0,061 = 0,044x + 0,008
0,053 = 0,044x
X = 1,20
2. Yes, because biuret is one of the best way to determine the
content of protein of a solution. In base solution, Cu 2+ will
form a complex with peptide bond of a protein, so that
producing a purple color which can be identified with
spectronic UV-Vis at wavelength 520 nm. This absorbance
is directly compare with concentration of protein and
doesnt depend on the kind of protein because all of
protein basically have the same amount of peptide bond
per weigh unit.

L. References

Dorey and Draves. Spectrophotometric Determination of Total ProteinBiuret

Method.Conway: University of Central Arkansas
Rbown.2002.The Structure of Protein.
(http://www.vivo.colostate.edu/hbooks/genetics/biotech/basics
/prostruct.html Accessed on November, 5th 2014)
Institute of Medicine.2005.Dietary Reference Intakes for Energy,
Carbohydrate, Fiber, Fat, Fatty Acids, Cholesterol, Protein,
and Amino
Acids (Macronutrients).Washington DC: National Academies
Press
Tim.2014.Petunjuk Praktikum Biokimia.Surabaya: FMIPA UNESA