DETERMINATION OF PROTEIN CONTENT

SPECTROPHOTOMETRICALLY
Name
NIM
Major/Study Program
Group
Date of lab work
The lab work name

: Tsabit Albanani
: 4301413003
: Chemistry/Chemistry Education
:7
: 15th September 2015
: Determination of Protein Content Spectrophotometrically

A. Objection
1. Understand the use of spectrophotometry as a tool to analyze the protein contents.
2. Explain the basic principles of the use of spectrophotometryin the analysis of
protein content
3. Skilfully use of spectrophotometry to determine of protein content.
B. Basic Theory
Protein is a high molecular organic compounds ranging from a few thousand to
millions. This protein is composed of atoms C, H, O and N, and other elements such as
P and S which form the amino acid units. The sequences of the amino acid sequence
with another amino acid, determining the biological properties of a protein. In nature
found a wide 20-21 amino build proteins. Protein is a vital nutrient group. This
compound is a protein found in the cytoplasm in living cells, both plant and animal. At
the protein chemically is hetero-polymer of amino acids bound together by peptide
bonds. Any protein and derived from any living creature also was only composed of 20
kinds of amino acids alone. Protein differences with each other due to the number and
position of the amino acids that have common characteristics as configuration 1, which
both have 1 COOH groups and one group CH2yang bound to the atom Ca (Lehinger,
2001).
When amino acids are linked together by acidamide bonds, linear
macromolecules (peptides) are produced. Those containing more than ca. 100 amino
acid residues are described as proteins (polypeptides). Every organismcontains
thousands of different proteins, which have a variety of functions. At a magnification of
ca. 1.5 million, the semischematic illustration shows the structures of a few intra and
extracellular proteins, giving an impression of their variety. The functions of proteins
can be classified as follows (Koolman and Roehm, 2005).
The structural proteins give extracellular structures mechanical stability, and are
involved in the structure of the cytoskeleton. Most of these proteins contain a high
percentage of specific secondary structures. For this reason, the amino acid
composition of many structural proteins is also characteristic.

Picture 1. Peptide bond and resonance in protein (Koolman and Roehm, 2005)
Measurement of protein content by Lowry method is the basic use of a
spectrophotometer. This method can use a protein content of up to 5 Micrograms. The
blue color that occurs by Folin reagent ciacalteu caused by the reaction between the
protein with Cu in solution and reaction alkaline phosphotungstate salt and salt
phosphomolybdat by tyrosine and tryptophan (Poedjiadi, 1994)
The wavelength controller (on the top of the instrument) adjusts the position of
the diffraction grating so that different colors of light are selected by the slit. The zero
% T control (on the left side of the front panel of the instrument) adjusts the
electronics. With the sample compartment cover down and no sample in the cell holder,
the zero control should be adjusted until the meter reads 0 for transmittance. This
control adjusts for the so-called dark current, the current transmitted by the photocell in
the dark. The 100%T control (on the right side of the front panel of the instrument)
adjusts the amount of light output while the blank cuvette is in the cell holder.
A mechanical linkage is used to drive a wedge into the light beam for this
adjustment. After inserting the blank cuvette into the cell holder, this knob should be
adjusted until the transmittance is 100% (or the absorbance is zero). This allows you to
correct for any stray absorbance resulting from the solvent alone.
A spectrophotometric method can be developed if the intensity of the coloration
produced is essentially dependent upon the concentration of the desired constituent,
and is stable long enough to permit measurement. The development of specific organic
reagents and effective means of complexation have made possible the determination of
single components found in complex systems. Furthermore, chemical phenomena
which occur in solution, as complex formation, solvation, and equilibria, may be
studied in situ by spectrophotometric methods. Absorption spectrophotometry thus
provides a useful analytical and research tool which bridges the span between the data
on atomic and ionic species supplied by emission spectroscopy and the information on
molecular and crystalline states obtained from X-ray diffraction (Timma, 1952)

C. Equipment and Materials

Equipment :
1. Spectronic 20
2. Cuvette
3. Pipette
4. Test tube
5. Erlenmeyer
6. Pumpkin peck
12. Materials :
1. Sample of Protein
2. CuSO4.5H2O
3. Sodium Potassium Tartrate
4. Aquades
5. NaOH 10%

7. Pipette volume
8. Ball pipette
9. Beaker glass
10. Volumetrick Flask
11. Test tube rack

6. Serum Albumin Pure

7. Casein
8. NaOH 3%
9.
10.

11.
D. Procedure
12. Experiment 1: Making Biuret Reagent
13.

dissolve 1,5 grams of CuSO4 and 6,0

grams of sodium potassium tatrate
(NaKC4O6.4H2O) in approximately
500 ml of distilled wate in a 1 liter
measuring flask

Add 300 ml of 10% NaOH

with a whipped-cream

Add distilled water to the line

14.
15. Experiment 2. Preparation of Standarf Solution Protein
16.

make a solution of pure serum

albumin in distilled water

Observe and note the

product

the yield of about 10 mg/ml

to facilitate solubility add a few

drops 3% of NaOH

17.
18.
19.
20.
21.
22. Experiment 3. Preparation of Calibration Curve
23.

24.

make a calibration curve

based on spectronik 20 data
on a standard protein
solution

Prepare a standart solution with a

concentration of 1 mg/ml protein, 3
mg/ml, 5 mg/ml, 7 mg/ml, and 9
mg/ml

use the blank solution containing

a mixture of 1 ml of distilled
water and 4 ml of biuret were
silenced 30 mimutes at room
temperature

insert standard solution into a test

tube 5 pieces (eat 1 ml). add 4 ml
of biuret reagent

shake and let sand for 30 minutes at

room temperature

enter into the cuvette, measure the

absorbance using spectronic 20 at a
wavelength of 540 nm

25.
26. Experiment 4. Measurement of Protin Samples
27.

Prepar the protein sample to be

analyzed

Add 1 ml of the sample into a

clean test tube, add 4 ml of
biuret reagent

shake and let stand for 30

minutes at room temperature

28.
29.
30.

use the calibration curve obtained to

calculate the protein content in the
sample

use the blank solution as at the

time of calibration curve

enter into the cuvette, measure

the absorbance using spectronic
20 at wavelength 540 nm

31.
32.
33.
34.
E. Result of Observation
35. Table 1. Result of observation
37.
Concen
36.
Name of
tration
38.
Absorbance
Test tube
(mg/ml)
39.
1
40.
1
41.
0,31
42.
2
43.
3
44.
0,16
45.
3
46.
5
47.
0,25
48.
4
49.
7
50.
0,34
51.
5
52.
9
53.
0,41
54.
Blank
55.
56.
0,00
57.
Sample of
58.
59.
0,40
Protein
60.

calibration curve
0.45
0.4

f(x) = 0.03x + 0.11

R = 0.6

0.35
0.3

Absorbance

0.25
0.2
0.15
0.1
0.05
0

10

times

61.
62.Graph 1. Relation between concentation protein and absorbance
63.
64.
F. Data Analysis and Explanation
65. In this experiment, purposed to determine the level of a protein in a solution
by using biuret reagent. In this experiment used a standard protein sample and random
sample solutions. The protein that used in these experiments are Albumin is by

dissolving 1 gram of albumin into 100 mL of distilled water. In this experiment used a
protein solution with a concentration that is different, that of 1, 3, 5, 7 and 9 mg / ml.
66. After each standard solution was put into a test tube and add biuret reagent
solutions then is left for 20 minutes. It is intended that the process of formation of
colored complex compounds can take place with absolutely perfect. After the colored
complex compounds are formed, then measured it

with the UV spectrometer.

Measurements were taken at a wavelength of 540 nm. In the spectrophotometer,

protein solution adsorbs light that given to it. This is an appearance of the interaction of
an atom with light. In which the electromagnetic energy is transferred to the atoms or
molecules so that the particles in protein promoted from a lower energy level to a
higher energy level, namely the level of excited. From the results of the identification
of the spectrophotometer, obtains of price absorbance at each concentration. The
greater the concentration, the more protein is absorbed, so the price the greater the
absorbance obtained as well.
67. Complex compounds is seen immediately after the addition of reagent
biuret with formation of a purple color in the solution. To get the color, then the protein
solution is treated with copper elements in the Biuret reagent. So we get a purple
protein solution at each concentration. The purple color is formed by the reaction
between Cu2+ in biuret reagent by peptide bonds of proteins in solution casein earlier,
precisely a bond with -NH of proteins as in the equation.
68.

69. From the results of the identification of the spectrophotometer, the price
obtained absorbance at each concentration as shown in the table. The greater the
concentration, the more protein is absorbed or absorbed, so the price the greater the
absorbance obtained as well.
70. Calculation:

71. R2
= 0,6011
72. y
= 0,0326x +0,109
73. concentration of sample protein:
74.
y
= 0,0326x +0,109
75.
0,40
= 0,0326x +0,109
76.
x
= 8,926 (the correct answer is 8)
result of observationthe correct answer
77. Percentage of error =
the correct answer
8,9268,000
8,000

78.

79.

= 11,5 %

x 100%

x 100%

80.
81.
82. Prior to testing samples of proteins, first attempted to create a calibration
curve. Measurement calibration curve was made by measuring levels of a protein in the
albumin with various concentrations is 1 mg/ml, 3 mg/ml, 5 mg/ml, 7 mg/mL and 9
mg/ml. Then measured absorbance at a wavelength of 540 nm which is the wavelength
of maximum. Absorbance value is directly proportional to the concentration. The
higher the absorbance value, the higher the concentration.
83. Based on the calibration curve, obtained by the equation y = 0,0326x
+0,109. Once the sample is obtained absorbance of the sample is measured so that the
protein content was obtained is 8,926 mg/ml. This result protein content of the sample
is 8 mg/ml. This experiment can conclusion that this experiment is correct, because
experiment error is 11,5 %.
84.
G. Conclusion and Suggestion
85. Conclusion
1. The higher the concentration of protein contained, more concentrated solution also
produced a purple color complex.
2. Absorbance of the solution is directly proportional to the concentration, so that the
greater the concentration used, the greater the absorbance used.
3. Concentration of random sample obtained is 8.926 mg/ml.
86. Suggestion
1. Try to understand the material before doing the lab work
2. We should use safety equipment when we are doing experiment.
3. Careful in use spectronic 20.
4. Dont forget to give label on test tube, so the test not will get accidently exchanged.

87.
H. Refferences
88.
Gilbert, Hiram F. 2000. Basic Concepts In Biochemistry. Texas: Baylor
College of Medicine
89.
Koolman, J and Roehm K.H. 2005. Color Atlas of Biochemistry. Germany:
Philipps University Marburg and Institute of Physiologic Chemistry
90.
Lehninger, Albert. 1995. Dasar-dasar Biokimia Jilid 1. Jakarta: Erlangga.
91.
Poedjiadi, A. 1994. Dasar-dasar Biokimia. Jakarta: UI-Press.
92.
Timma, D.L. 1952. Absorption Spectrophotometry. Columbus: Ohio Journal of
Science
93.
94.
Semarang, 18th October 2015
95. Lab Work Lecturer
Student
96.
97.
98.
99. Samuel Budi W.K M.Si, M.Sc
Tsabit Albanani
100.NIP 198204182006041002
NIM 4301413003
101.
102.
103.
104.Question
1. Explain the legal basic of the analysis using spectrophotometry tool
2. Describe the work of spectrophotometry instrument. Describe the work of visible
spectrophotometric instruments
3. Explain the advantaged and disadvantages of the use of the biuret method with
analysis spectronic 20 for protein content
4. Why the reaction in this experiment is called biuret reaction ?
105.
106.Answer
1. The underlying law is the law of Lambert-beer:
107."Measuring the intensity of monochromatic light through a colored solution that
lasts exponentially and depends on the wavelength of the light passed through the
solution and the concentration of substances in solution"
I
Io

108.

A = log

= - KCL

109.
110.
111.
112.
113.

Description:
I
= intensity of the light
Io
= the intensity of the light emitted
K
= concentration based on the nature of the substance in solution
C
= concentration of solute

114. L
= length of light through which the solution
115.
light
light
light
2. S
D
L
be continued
monochro
monochro
Light
of
116.
117. Description:
118. S
= light source
119. D
= sample/solution
120.L
= prism
3. Pros: Reagents biuret more durable in storage, showed a positive reaction to a bully
biuret compounds.
121.Disadvantages: the bully biuret compounds
4. The reaction of this experiment called biuret reaction because it gives a positive
reaction to biuret.
122.

123.
124.