DEPARTMENT
OF BIOLOGY
FACULTY
OF SCIENCE & MATHEMATICS
UNIVERSITI
PENDIDIKAN SULTAN IDRIS
SBK3013
PRINCIPLE
IN BIOCHEMISTRY
EXPERIMENT 5: PROTIEN (AMENDED)
NAME
|
MATRIC NO.
|
MUHAMMAD FARIS BIN ISMAIL SAZEMI
|
D20141067089
|
MAYURIE PHUTHARANT A/P SURIN
|
D20141067078
|
NUR AFIQAH SYAHMINA BT MOHD KAMAL
|
D20141067091
|
GROUP: A
LECTURER’S NAME: DR. ROSMILAH MISNAN
INSTRUCTOR NAME: NUR ATIEKAH BT AZAHARI
INTRODUCTION
Determining
the exact quantity of proteins in a solution is very often necessary in the
biochemical practice. There are many ways to measure protein concentration. In
chromogenic methods, the absorbance of a coloured product formed by the protein
and an organic molecule is measured. Protein concentration can also be
determined from the protein’s UV absorbance. However, these methods may give
different results for different proteins of the same concentration. Different
methods also can yield different results for the same protein. There is no
absolute photometric protein concentration assay. All methods have advantages
and disadvantages and we must choose among them by taking the following aspects
into consideration: specificity, sensitivity, the measurable range of
concentration, the accuracy, the nature of the protein to be examined, the
presence of materials interfering with the measurement, and the time required
for the measurement.
The
first test is to determine the protein concentration by using the Biuret assay
method. Molecules with two or more peptide bonds react with Cu2+
ions in alkaline solution and form a purple complex. Nitrogen atoms of the
peptide bonds form a coordination bond with the metal ion. The quantity of the
complexes formed is proportional to the number of peptide bonds. In practice,
the determination of protein concentration is done using a calibration curve
created using samples of known concentration. The protein treated with biuret
reagent is measured at 540 nm after the purple product is formed. The
advantages of the method include that only few materials (e.g. Tris and amino
acid buffers) interfere with it, it can be done in a short time and does not
depend on the amino acid composition of the protein. Its disadvantages are its
low sensitivity and that it requires at least 1 mg of protein.
Second
test to determine the protein concentration is by using the Lowry assay method.
The advantage of this method is Lowry assay is sensitive to low concentrations
of protein. The major disadvantage of the Lowry method is the narrow pH range
within which it is accurate. A variety of compounds will interfere with the
Lowry procedure. These include some amino acid derivatives, certain buffers,
drugs, lipids, sugars, salts, nucleic acids and sulphydryl reagents. The ammonium
ions, zwitter ionic buffers, nonionic buffers and thiol compounds may also
interfere with the Lowry reaction. These substances should be removed or
diluted before running Lowry assays.
MATERIAL
·
Protein
standard
·
Biuret
reagent
·
Lowry
reagent
·
Sample:
fish, beef, chicken, peanut, green bean, soybean, red bean and dal bean
PROCEDURE
1.
Preparation of protein standard
1. Solution
of gelatin at 1, 2,3,4,5 and 6 mg/mL in water from the gelatin stock solution
(10 mg/mL) for biuret assay was prepared.
2. Solution
of gelatin at 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 mg/mL in water from the gelatin
stock solution (1 mg/mL) for Lowry method was prepared.
2.
Preparation of test samples
a. Animal
protein
1. 10
g of protein sample was weighted
2. Macerated
into smaller size
3. Phosphate
Buffer saline at 1:10 ratio was blended
4. Sample
was filtered by kitchen filter
5. The
supernatant was collected
6. Sample
was filtered again using Whatman filter No 1
7. The
supernatant was collected
b. Plant
protein
1. 10
g of protein sample was weighted
2. The
sample was crushed and grinded into fine paste or powder using mortar and
pestle.
3. The
powder was dissolved in Phosphate Buffer saline at 1:10 ratio
4. Sample
was filtered by kitchen filter
5. The
supernatant was collected
6. Sample
was filtered again using Whatman filter No 1
7. The
supernatant was collected
3.
Protein assay
a. Biuret
assay
1. 0.5
mL of each protein was mixed with 2.50 mL of biuret reagent
2. The
absorbance of the samples at 540 nm after 10 minutes was measured
3. The
standard curved was plotted
4. The
protein content of the test sample was estimated using the standard curve.
b. Lowry
assay
1. 0.25
mL of each protein was mixed with 2.5 mL of
Lowry reagent 1
2. The
mixture was incubated at room temperature for 10 minutes.
3. 0.25
mL of Lowry reagent 2 was added and mixed immediately
4. The
mixture was incubated at room temperature for 30 minutes.
5. The
absorbance of the samples at 750 nm was measured.
6. The
standard curved was plotted
7. The
protein content of the test sample was estimated using the standard curve.
RESULT
Protein Standard
1. Biuret
Protein Number
|
|
B1
|
0.439
|
B2
|
0.509
|
B3
|
0.512
|
B4
|
0.542
|
B5
|
0.769
|
B6
|
0.995
|
2. Lowry
Protein Number
|
|
L1
|
0.132
|
L2
|
0.106
|
L3
|
0.406
|
L4
|
0.256
|
L5
|
0.115
|
L6
|
0.081
|
Protein Number for Other Sample
Sample
|
Biuret
|
Lowry
|
Beef
|
0.488
|
0.944
|
Fish
|
1.020
|
1.320
|
Braise
Fish
|
1.138
|
0.406
|
Chicken
|
0.552
|
1.328
|
Soybean
|
0.447
|
0.377
|
Red
bean
|
0.645
|
0.926
|
Peanut
|
0.476
|
1.219
|
Dal
bean
|
0.746
|
0.214
|
Green
bean
|
0.983
|
1.945
|
DISCUSSION
In Biuret assay we
combine protein samples with Biuret Reagent which contains copper ions in a
basic solution. The copper ions will complex with the amide groups in the
proteins to create a blue colour that will be measured using a
spectrophotometer. The amount of blue colour that forms is directly
proportional to the quantity of protein in the sample. In order to determine
the actual concentration of protein in the unknown sample it is necessary to
graph the standard curve. Our standard graph is shown as above.
The Lowry assay combines the reactions of copper ions with the peptide
bonds under alkaline conditions (the Biuret test) with the oxidation of
aromatic protein residues. The Lowry method is used with protein concentrations
of 0.01–1.0 mg/Ml. It is based on the reaction of Cu+, produced by the
oxidation of peptide bonds, with Folin–Ciocalteu reagent. The concentration of
the reduced Folin reagent is measured by absorbance at 750 nm. As a result, the
total concentration of protein in the sample can be deduced from the concentration
of Trp and Tyr residues that reduce the Folin–Ciocalteu reagent.
In our experiment, the results for Biuret test showed that braise fish
has the highest protein number of 1.138 followed by fish, green bean, dal bean,
red bean, chicken, beef, peanut and soybean. In the other hand, Lowry test
showed that green bean has the highest protein number of 1.945 followed by
chicken, fish, peanut, beef, red bean, braise fish, soybean and dal bean.
There are few errors occur in our experiment. First and foremost, the
measuring cylinder that have been used to measure volume each samples are not
cleansed thoroughly, and this may due to some particles left can alter the
measurement of each sample. Furthermore, the absorbance of samples are measured
less than 10 minutes. This may cause the reading of the absorbance are not
accurate.
QUESTION AND ANSWER
1.
3 alternative methods of determining
protein concentration are
i)
By using UV absorbance. Protein concentrations can be determined directly by
ultraviolet spectroscopy because of the presence of tyrosine and tryptophan
which absorb at 280 nm. Furthermore, this method include high sensitivity so
valuable protein samples can be recovered.
ii)
BCA
Protein Assay. Bicinchoninic acid (BCA) reacts with
cuprous ions to generate purple colour at 562 nm. Cuprous ions are produced by
the reduction of cupric ions by proteins in alkaline solutions. It is
compatible with a large number of extraneous materials found in protein
preparations.
iii)
Dye-binding
method. Dye Coomassie Blue G-250 is dissolved in an acidic
solution causing it to absorb at 465 nm (reddish brown). When the dye
(negatively charged) binds to the positively charged protein molecule the
absorbance undergoes a shift to 595 nm (blue). This shift in absorption maximum
is proportional to protein concentration over a broad range.
2.
Appropriate blank is a reference
measurement of the transmitted light as a function of wavelengths is stored in
memory. When a measurement of a sample is made, the intensity of light that has
been transmitted through the sample is recorded. Appropriate blank is needed to
calibrate the spectrophotometer. This is because it may be some other particles
or substances presence in the sample.
CONCLUSION
In
conclusion, for the Biuret test, braise fish has the highest protein number
that is 1.138 meanwhile for the Lowry test, green beans shows the highest
protein number that is 1.945 when compared to the other sources of protein.
REFERENCES
N. A. Khan and K. N. Singh. 2014. Laboratory manual of
biochemistry. New Delhi: Daya
Pub. House
Pub. House
Moran, Laurence A., Horton, H. Robert, Scrimegeour, K. G. and Perry,
Marc. 2014. Principle
in Biochemistry. 5th ed. Upper Saddle River, NJ: Prentice Hall.
in Biochemistry. 5th ed. Upper Saddle River, NJ: Prentice Hall.
