A.

TITLE

: Determination The Percentage of Protein Through Biuret Method

B. DATE OF EXPERIMENT : November, 7th 2013

C. PURPOSE

: Determining the percentage protein of a sample using Biuret Method

D. BASIC THEORY :
Proteins are macromolecules with molar masses ranging upwards of 6000 Daltons.
They are formed from colvalently bonded amino acids units. The simplest amino acid is
glycine whose structure is:

Proteins are formed by the linkage of these amino acid groups via an amine
group-carboxylic acid group covalent bond which also produces water.

Hundreds to

thousands of amino acids are present in proteins. There are 20 standard amino acid groups
which vary only in what is called the side chain. That is, the group which is attached to
the central carbon (in glycine it is Hydrogen, in others it may be a methyl group or a phenyl
group or something else). It is the combination of these 20 amino acids and how they are
ordered which dictates the function of a protein.

Proteins form between 50 and 70 % of a cells dry weight and are found in all cells,
secretions, fluids and excretions of the body. The concentration of proteins in the body
ranges from 6.0 g/dL to 8.3 g/dL. The most abundant protein is albumin which can make up
60% of the total protein concentration.
Spectrophotometric analysis relies on the interaction of electromagnetic radiation
(light) with the matter of interest.

Strictly speaking, every compound has a distinct

absorption spectrum which allows its identification, in many cases, in the presence of other
compounds. In addition to the identification of a compound, it is also possible to determine
quantitatively the concentration of that compound. The relationship between absorbance
and concentration is given by the Lambert-Beer Law and is written mathematically as:

A=l c
Where A is the absorbance, a unit-less quantity;
is the molar absorptivity constant ( a constant of the compound having
units of L*mol-1*cm-1); and
l is the path length over which the light interacts with the sample in cm.

In a more practical sense, the absorbance is defined as the negative logarithm of the
transmittance This is given mathematically as:
A = - log T = - log
where I is the intensity of the light beam when the sample is present; and I0 is the intensity of
the light beam when everything but the sample is present. ( in some cases this is called a
background or a blank)

In analytical spectrophotometry, the molar absorptivity of the unknown compound

may not be known. It is therefore a common practice to generate a calibration or standard
curve. A standard curve is generated by measuring the absorbance of a series of samples for
which the concentration is known. Then, since the Lambert -Beer Law shows a linear
relationship between absorbance and concentration, a linear least squares fit of the standard
curve will yield a mathematical relationship between the absorbance and concentration.
This relationship in turn can them be used to calculate the concentration of the unknown
sample.

Biuret method is one of the best way to determine protein level of solution. In base
solution, Cu2+ forms complex with peptide bonding of protein. It will produce purple color
with maximum absorbance in 540 nm. Absorbance is equal with concentration of protein. It
doesnt depend in protein types, because all protein have same amount of peptide bonding
per weight.

This reaction can be disturbed by the existence of urea or compounds that contain CONH- groups, and reductor sugar that reacts with Cu2+.

In a positive test, a copper (II) ion is reduced to copper (I), which forms a
complex with the nitrogen and carbons of the peptide bonds in an alkaline solution. A violet
color indicates the presence of proteins.

It is possible to use the Biuret reaction to determine the concentration of proteins

because peptide bonds occur with approximately the same frequency per gram of material.
The intensity of the color, and hence the absorption at 540 nm, is directly proportional to the
protein concentration, according to the Beer-Lambert law.

The Biuret reagent is made of potassium hydroxide (KOH) and hydrated copper(II)
sulfate, together with potassium sodium tartrate. Potassium sodium tartrate is added to
complex and stabilize the cupric ions. The reagent turns from blue to violet in the presence
of proteins, blue to pink when combined with short-chain polypeptides. Not all biuret tests
require the biuret reagent. The reagent is commonly used in a biuret protein assay, a
colorimetric assay used to determine protein concentrationsuch as UV-VIS at wavelength
540 nm (to detect the Cu2+ ion).

UV-Visible spectro photometric, when a molecule absorbs UV/Vis radiation, it

undergoes a change in its valence shell configuration. Basic principles spectrometry analysis,
sample solution absorbs electromagnetic radiation and the intensity of radiation which
absorbed by the sample solution is connected with the concentration of analytes (substances
/elements to be analyzed) in the sample solution.

The visible spectrum with respect to infrared and ultraviolet radiation.

E. TOOLS AND MATERIALS

Tools :
Beaker glass 250 mL

(1 pieces)

Pipette

(10 pieces)

Test tube

(10 pieces)

Graduated cylinder 10 mL (2 pieces)

Volumetric flask 10 mL

(1 piece)

Pro pipette

(1 pieces)

Rack

(1 piece)

Materials :

Protein standard solution

Biuret reagent
Aquades
Sample solution of protein

F. PROCEDURE
Standard solution preparation
1 ml of rotein
standard
solution 1mg/ml

1 ml of rotein
standard solution
2 mg/ml

1 ml of rotein
standard
solution 3 mg/ml

Result

Result

Result

1 ml of rotein
standard solution
4 mg/ml

1 ml of rotein
standard solution
5 mg/ml

Poured into test tube

Added mL Biuret reagent
shaked
incubated in 37 for 10 15 minutes or
in room temperature 30 minutes until the
purple color stable
determined the absorbance by using UV
VIS spectrophotometric 520 nm

Result

Result

Absorbance determination of blanco solution

1 ml of aquades

Poured into test tube

Added mL Biuret reagent
shaked
incubated in 37 for 10 15 minutes or
in room temperature 30 minutes until the
purple color stable
determined the absorbance by using UV
VIS spectrophotometric 520 nm

Result

Absorbance determination of sample solution

1 ml of sample
-

Result

Poured into test tube

Added mL Biuret reagent
shaked
incubated in 37 for 10 15 minutes or
in room temperature 30 minutes until the
purple color stable
determined the absorbance by using UV
VIS spectrophotometric 520 nm
repeated 3 times

G. TABLE OF DATA
No
1.

Procedure
Standard solution preparation
1 ml of rotein standard
solution 1mg/ml
-

Poured into test tube

Added mL Biuret reagent
shaked
incubated in 37 for 10 15 minutes or in
room temperature 30 minutes until the
purple color stable
determined the absorbance by using UV
VIS spectrophotometric 520 nm

Result
1 ml of rotein standard
solution 2 mg/ml
-

Poured into test tube

Added mL Biuret reagent
shaked
incubated in 37 for 10 15 minutes or in
room temperature 30 minutes until the
purple color stable
determined the absorbance by using UV
VIS spectrophotometric 520 nm

Result
Before
Biuret reagent : blue
Protein standard :
colorless

Hypothesis

Potassium sodium tartrate is

The higher
concentration of
sample the higher the
absorbance, it because
there are many protein
already react with
biuret reagent.
The equation is
y =0.0517x + 0.034

added to complex and

R = 0.9333

The Biuret reagent is made of

potassium hydroxide (KOH)
and hydrated copper (II)
sulfate, together with
potassium sodium tartrate.

After
Buret + protein
standard
1ppm : purplish blue
(+)
2ppm : purplish blue
(++)
3ppm : purplish blue
(+++)
4ppm : purplish blue
(++++)
5ppm : purplish blue
(+++++)

stabilize the cupric ions. The

reagent turns from blue to
violet in the presence of
proteins, blue to pink when
combined with short-chain
polypeptides.

Protein + NaOH + CuSO4 +

NaK tartat

+ KOH + Na2SO4
Result

Conclusion

1 ml of rotein standard
solution 3 mg/ml
-

Poured into test tube

Added mL Biuret reagent
shaked
incubated in 37 for 10 15 minutes or in
room temperature 30 minutes until the
purple color stable
determined the absorbance by using UV
VIS spectrophotometric 520 nm

Absorbance
1 ppm : 0.096
2 ppm : 0.164
3 ppm : 0.215
4 ppm : 0.239
5 ppm : 0.266

y =0.0517x + 0.034

Result

R = 0.9333
1 ml of rotein standard
solution 4 mg/ml
-

Result

Poured into test tube

Added mL Biuret reagent
shaked
incubated in 37 for 10 15 minutes or in
room temperature 30 minutes until the
purple color stable
determined the absorbance by using UV
VIS spectrophotometric 520 nm

1 ml of rotein standard
solution 5 mg/ml
-

Poured into test tube

Added mL Biuret reagent
shaked
incubated in 37 for 10 15 minutes or in
room temperature 30 minutes until the
purple color stable
determined the absorbance by using UV
VIS spectrophotometric 520 nm

Result

Absorbance determination of blanco solution

1 ml of aqudes
-

Result

Poured into test tube

Added mL Biuret reagent
shaked
incubated in 37 for 10 15 minutes or in
room temperature 30 minutes until the
purple color stable
determined the absorbance by using UV
VIS spectrophotometric 520 nm

Before
Aquadest = colorless
Reagent biuret = blue
solution
After
Aquadest + Biuret =
blue

It is used as reduction factor to The color of biuret is

blue, it means it didnt
calculate the absorbance of
contain protein
protein+biuret complex in UV
spectrofotometri, as well to
compare the color of
containing and not-containing
protein

Absorbance determination of sample solution

1 ml of sample
-

Result

Poured into test tube

Added mL Biuret reagent
shaked
incubated in 37 for 10 15 minutes or in
room temperature 30 minutes until the
purple color stable
determined the absorbance by using UV
VIS spectrophotometric 520 nm
repeated 3 times

Before
Sample = colorless
Biuret = blue
After
Sample + biuret =
purplish blue
Absorbance of sample:
A1 : 0.056
A2 : 0.057
A3 : 0.057
Concentration of:
Sample 1 : 0.4255
Sample 2 : 0.4448
Sample 3 : 0.4448
The average
concentration : 0.4383

Color changes to light purple

or purplish blue after adding
biuret method, indicates the
existences of peptide bond or
protein in the sample

The color of sample

after added by biuret is
purplish blue, means
the sample contain
protein. From
calculation of UV VIS
spectroscopy result, the
concentration of
sample is 0.4383
mg/ml

H. ANALYSIS OF DATA
The purpose of this experiment is determining the percentage protein of a sample using
Biuret Method. Biuret method is one of the best way to determine protein level of
solution. In base solution, Cu2+ forms complex with peptide bonding of protein. It will
produce purple color with maximum absorbance in 540 nm.

Preparation of blanco solution

1 ml of aquades added with 4 ml of biuret reagent. Test tube then closed with aluminum
foil to prevent from the air contamination and heat in water bath 37C for 10 minutes to
form a stable purple complex color. After 10 minutes, there is no changes of blanco
solution color (still blue). It indicates that the solution contains no peptide chain (no
protein found in solution) since it gives negative result with biuret reagent.

Determining the absorbance of sample solution

Three sample solution is poured 1 ml in each 3 test tubes. Then these sample solution was
added with 4 ml of biuret reagent, closed with aluminum foil, heat in water bath for 10
minutes minutes to form a stable violet complex color, to make the color more visible.
The color changes to purplish blue, indicates the existences of peptide bond or protein in
the sample. Then absorbance of all sample solution is measured using UV
spectrophotometry. From the measurement the absorbance of sample are:
No

Sample

Absorbance

1.

Sample 1

0.056

2.

Sample 2

0.057

3.

Sample 3

0.0577

Determining the absorbance of standard solution

In this experiment five standard solutions with different concentrations was made and
measured the absorbace. The standard solution made by dilution. To make this standard
solution, the mother liquor is diluted according the desired concentration. Mother
solution having a concentration of 10 mg / mL. The color of mother liquor is colorless.
Concentration of standard solution required is 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg /

mL and 5 mg / mL. To obtain standard solutions with different concentrations, we dilute

solution of 10 mg / mL by using the following formula:
M1V1 = M2 V2
where :
M1 = concentration of mother liquor
V1 = volume of mother liquor
M2 = concentration of standard solution
V2 = volume of volumetric flask
From that equation, the volume of mother liquor to prepare standard solution 1 mg / mL,
2 mg / mL, 3 mg / mL, 4 mg / mL and 5 mg / mL are 1 ml, 2 ml, 3 ml, 4 ml and 5ml. 1
ml of This standard solution then poured in test tube and given label. Then added with 4
ml of biuret reagent. Test tube then closed with aluminum foil to prevent from the air
contamination and heat in water bath 37C for 10 minutes to form a stable purple
complex color. After 10 minutes, the blue solution from each solution becomes purple.
The color of each standard solution shown by the following table:
Standard solution

Color

1 ppm

Purplish blue (+)

2 ppm

Purplish blue (++)

3 ppm

Purplish blue (+++)

4 ppm

Purplish blue (++++)

5 ppm

Purplish blue (+++++)

Biuret reaction is a common color reaction for peptide group (-CO-NH-) and
protein. The positive reaction is characterized by the formation of a purple color as in the
solution of complex compounds formed between Cu

2+

ions and peptide bonds N of

molecules that takes place under alkaline conditions. The number of amino acids bound
to the peptide bond affects the color reaction. It is appropriate with theory; the more
concentrated the solution, the more violet the color. It indicates the existences of protein
in the solution. Then absorbance of all standard solution is measured using UV
spectrophotometry. UV-Visible spectro photometric, when a molecule absorbs UV/Vis
radiation, it undergoes a change in its valence shell configuration. Basic principles

spectrometry analysis, sample solution absorbs electromagnetic radiation and the

intensity of radiation which absorbed by the sample solution is connected with the
concentration of analytes (substances /elements to be analyzed) in the sample solution.
Thats why firstly we test the blanco solution as the comparison to compare the color of
containing and not-containing protein. We know that blanco solution contains no protein
since it gives negative result (theres no color change). From the measurement, the
absorbance of standard as follow :
No

Concentration

Absorbance

1.

1.000

0.096

2.

2.000

0.164

3.

3.000

0.215

4.

4.000

0.239

5.

5.000

0.266

The correlation coefficient is 0,95026

Determining the concentration of sample

A standard curve is generated by measuring the absorbance of a series of samples
for which the concentration is known. Then, since the Lambert -Beer Law shows a linear
relationship between absorbance and concentration, a linear least squares fit of the
standard curve will yield a mathematical relationship between the absorbance and
concentration. This relationship in turn can them be used to calculate the concentration of
the unknown sample. The linear relationship between absorbance and concentration of
our experiment as follow :

Standart Curve
0.35
0.3

y = 0.0517x + 0.034
R = 0.9333

absorbance

0.25
0.2

Absorbance

0.15

Linear (Absorbance)

0.1
0.05
0
0

concentration

A mathematical relationship between the absorbance and concentration from that curve is
y = 0.0517x + 0.034
R = 0.9333
This relationship in turn can be used to calculate the concentration of the unknown sample.
The concentration of sample shown by x while y is the absorbance of sample. From the
calculation, the concentration of sample are:
No

Sample

Absorbance

Concentration

1.

Sample 1

0.056

0.4255

2.

Sample 2

0.057

0.4448

3.

Sample 3

0.057

0.4448

Average

0.4383

In our calculation result, we got the concentration of sample is 0.4383. From this result we
can know that the percentage of protein in sample solution is very low, lower that the
percentage of protein that contain in the lowest standard solution.

I. CONCLUSION
The percentage protein of sample is very low, lower than percentage of the lowest
standard solution, that is only has concentration 0.4383 mg/ml

J. REFERENCES
Anonymous.Spectrophotometric Determination of Total Protein-Biuret Method.
(Online). http://www1.gantep.edu.tr/~balci/protein%20%2816%29.pdf
(accessed on November,4th 2013)
Aldhini. 2012. Uji Biuret (Online). http://aldhini.blogspot.com . (Accessed on 06
November 2013)
Lehninger, Albert L. 1982. Principle of Biochemistry (Dasar-DasarBiokimia,
AlihbahasaMaggyThenawidjaja). Jakarta: Erlangga
Tim Dosen Biokimia. 2013. PetunjukPraktikumBiokimia. Surabaya: Unesa
University Press.

ANSWER QUESTION
1. Make standard curve concentration vs absorbance. With those standard curve
determining percentage of protein sample !

Standart Curve
0.35
0.3

y = 0.0517x + 0.034
R = 0.9333

absorbance

0.25
0.2

Absorbance

0.15

Linear (Absorbance)

0.1
0.05
0
0

concentration

A mathematical relationship between the absorbance and concentration from that curve
is y = 0.0517x + 0.034 and R = 0.9333

Percentage of sample :
No

Sample

Absorbance

Concentration

1.

Sample 1

0.056

0.4255

2.

Sample 2

0.057

0.4448

3.

Sample 3

0.057

0.4448

Average

So the percentage of sample : 0.4383 mg/ml

0.4383

2. Is peptide will give positive reaction toward biuret reagent? If it is true, how to
determine percentage of protein that mixed with peptide ?
Answer :
Peptide will give positive reaction toward biuret reagent, it proved with
presence of violet blue (purple) color. It occur when Cu2+ ion form complex with
peptide bond in a protein solution.
To determine percentage of protein that mixed in peptide is by measuring
adsorance of sample with spectrophotometer of UV-Vis. Measuring with
spectrophotometer is depend on measuring of matter interaction with
electromagnetic radiation. In measuring with spectrophotometer UV-Vis, the
color of each solution have certain wave length so to determine percentage of
protein that mixed with peptide using standard solution to determine the
equation y = ax+b appropriate with Lambert-Beer law where y is absorbance of
measured protein, a is coefficient, x is percentage of protein that searched, b is
slope that produce by graphic.

ATTACHMENT

No.

Picture

Explanation

Biuret reagent. The color of

boiret reagent is blue

Standar, sample and blanco

solution incubated and
observed until the color is
stable

Blanco solution still blue,

indicated thre isnt contain
protein

The standard solution

( from left to right) 5ppm,
4ppm, 3ppm, 2 ppm, 1ppm.
The color of solution from
left to right was lighter. It
indicated that the
percentage of protein in
solution was smaller

Standars, sample and blanco

solution was measured with
UV VIS spectroscopy