TITLE
Experiment 2: Protein Experiment
OBJECTIVES
1. To
determine protein concentration in various type of protein content
2. To
determine protein concentration using two types protein assays that are Biuret
assay and Lowry assay.
INTRODUCTION
In the experiment to determine the
protein concentration, Biuret assay and Lowry assay is used.The biuret test is a chemical test used for detecting the
presence of peptide bonds. In the presence of peptides, a copper(II) ion forms violet-coloredcoordination
complexes in an alkaline solution. Several variants on the test have been
developed, such as the BCA test and the Modified Lowry test. The Biuret
reaction can be used to assess the concentration of proteins because peptide
bonds occur with the same frequency per amino acid in the peptide. 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. Despite its name, the reagent does
not in fact contain biuret ((H2N-CO-)2NH).
The test is so named because it also gives a positive reaction to the
peptide-like bonds in the biuret molecule.
While, the Lowry protein assay is a biochemical assay for determining the total level of protein in a solution. The total protein concentration is exhibited by a color change of the sample solution in proportion to protein concentration, which can then be measured using colorimetric techniques. It is named for the biochemist Oliver H. Lowry who developed the reagent in the 1940s. His 1951 paper describing the technique is the most-highly cited paper ever in the scientific literature, cited over 200,000 times. The method 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 best used with protein concentrations of 0.01–1.0 mg/mL. Lowry test is measured for the absorbance at 750 nm.
PROCEDURE
Dilution
1. Protein
is diluted for 10% and sample is diluted for 100% in order to make sure the
validity of protein absorbance for protein test.
2. Protein
with 5 different volume that are 1mg/mL, 2 mg/mL, 3mg/mL, 4mg/mL, 5mg/mL and
6mg/mL is diluted for 10% by mixing 1mL of protein with 9mL of distilled water.
3. Protein
sample with 5 different protein sample that are duck egg, chicken egg, omega
egg, puyuh egg, and ayam kampung egg firstly is diluted for 10% by mixing 1ml
of protein sample with 9mL of distilled water.
4. The
diluted protein sample is then diluted for another 10% by mixing 1 ml diluted
protein sample from the first dilution with 9ml of distilled water.
a
Biuret assay
1.
The experiment is done with
5 different 10% diluted protein that are 1mg/mL, 2 mg/mL, 3mg/mL,
4mg/mL, 5mg/mL and 6mg/mL .
2. The
experiment also repeated by replacing protein with 5 different protein sample
that have been diluted for 100 % that are duck egg, chicken egg, omega egg,
puyuh egg, and ayam kampung egg.
b Lowry assay
RESULT
1. Standard Protein
Standard protein (mg/ml)
|
Absorbance (nm)
|
1
|
0.155
|
2
|
0.193
|
3
|
0.210
|
4
|
0.273
|
5
|
0.317
|
6
|
0.357
|
2. Samples Protein
Types of sample egg
|
Absorbance based on graph (nm)
|
Protein sample (mg/ml)
|
Duck
|
0.273
|
4.00
|
Chicken 1 (Normal Chicken)
|
0.331
|
5.35
|
Chicken 2 (Ayam
Kampung)
|
0.298
|
4.50
|
Omega 3
|
0.214
|
2.60
|
Quail (Puyuh)
|
0.253
|
3.50
|
Graph and Calculation
Full view
Larger view
A) Biuret assays
Dilution of the
solution samples:
The actual
concentration of the protein in the samples:
a)
Omega 3
= 2.6 mg/ml × 50
times of dilution
= 175 mg/ml
b)
Quail
= 3.5 mg/ml × 50
times of dilution
= 175 mg/ml
c)
Duck
= 4.0 mg/ml × 50
times of dilution
= 200 mg/ml
d)
Chicken 1 (ayam biasa)
= 4.5 mg/ml × 50
times of dilution
= 225 mg/ml
e)
Chicken 2 (ayam
kampung)
= 5.35 mg/ml × 50
times of dilution
= 267.5 mg/ml
DISCUSSION
The subunits which
make up proteins are amino acids. The amino acids are joined together by
dehydration synthesis to forms chains, which are hundreds of amino acids long
which is called proteins. Proteins function as enzymes or as structural units
in cells. They do most of the work in a cell. Almost all of the exciting stuff
such as metabolism, memory, hormone action, and movement involves proteins. In
this lab, we have learnt method of measuring protein concentration, biuret
assay.
The biuret reaction is a method that can be used to determine the amount of soluble protein in a solution. The biuret reagent (copper sulfate in a strong base) reacts with peptide bonds (which join amino acids to form proteins) and changes colour when it does so. The spectrophotometer has been used to measure the intensity of the colour produced. The more protein present the darker the colour.
In order to quantitatively determine how much protein is represented by a particular absorbance reading it is necessary to construct a standard curve. This is done by performing the biuret reaction on a series of prepared solutions of gelatin at 1,2,3,4,5 and 6 mg/ml in water. The absorbance readings obtained from these solutions are used to construct a graph of absorbance as a function of protein concentration. This graph is called the standard curve for assay, and can be used to convert the absorbance readings for the experimental samples (eggs from duck, chicken 1 (ayam biasa), chicken 2 (ayam kampung), omega 3, quail) into a protein amount or concentration.
Based on the graph that has been constructed, it shows that the standard protein concentration for the samples as shown in the result. From the graph we can see that, the highest concentration of protein is chicken 1 and the lowest is omega 3. But for the real result actually the omega 3 will get the highest and the chicken1 will get the lowest. So, from the experiment maybe there are some mistake happen while conducted it. That’s why our group get the opposite result than the actual one. Error that occurred could be the mixing of using the same pipette.
Lowry method is
one of the ways to determine the protein concentration in a solution. However
it needs two-step procedure which requires a minimum of 40 minutes incubation
time. The sensitivity of Lowry Assay reagent is greatly enhanced over that of
the Biuret total protein reagent. The working range of the method covers the
total protein rage from 1 to 1500 μg/mL. However, the working range for Biuret
assay is from 5 to 160 mg/mL. The reading of colour at 750 nm for Lowry Assay
is recommended because at this wavelength, few other substances absorb light.
The amount of light absorbed at 750 nm is directly proportional to the amount
of protein in the sample.
CONCLUSION
Throughout
this lab, we can conclude that there are two ways of measuring protein
concentration in a solution. It is either by using biuret assay or Lowry assay.
Both can be used to measure the protein
concentration in a solution but the most accurate way to measure the
concentration is by using Lowry assay. This is due to the fact that Lowry assay
can measure the protein from as low as 1 μg/mL to 1500 μg/mL compared to biuret
which can only measure the protein from 5 to 160 mg/mL. in this experiment, we
had tested the protein concentration in five different types of eggs, which
are, duck’s egg, ayam biasa ( chicken
1 ) and ayam kampung ( chicken 2 ),
omega 3 egg and quail’s egg. From the graph plotted, chicken 1 contain the most
protein while omega 3 contain the least. The results supposed to be vice versa,
omega 3 should contain the most protein whereas chicken 1 the least. This
happened due to some mistakes we make during pipetting the samples.
REFERENCES
