Protein standard
Biuret reagent
Lowry reagent 1 and
Lowry reagent 2
8 Test samples:
a.
Animal samples: chicken, fish, beef, boiled fish
b.
Plant proteins: Soybean, red bean, peanut, dhal bean
PROCEDURE
1.
Preparation of Protein Standard
1. Solutions 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. Solutions 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 gram of protein
samples was weighted.
2. The sample was macerated
into smaller size.
3. Sample was blend in
Phosphate Buffer saline at 1:10 ratio.
4. The sample was shake/
stir/ for 15 minutes.
5. Sample was filtered
by kitchen filter.
6. The supernatant was
collected.
7. Sample was filtered
again using Whatman filter No 1.
8. The supernatant was
collected.
b) Plant Protein
1. 10 gram of protein
samples was weighted.
2. The sample was crush
and grind into a fine paste or powder using mortar and pestle.
3. The powder of sample
was dissolve in Phosphate Buffer saline at 1:10 ratio.
4. The sample was
shake/ stir/ for 15 minutes.
5. Sample was filtered
by kitchen filter.
6. The supernatant was
collected.
7. Sample was filtered
again using Whatman filter No 1.
8. The supernatant was
collected.
3.
Protein Assay
a) Biuret assay:
1. All 8 test sample
extracts was obtained from the other groups.
2. 0.50 mL of each
protein (standard and test samples) was mixed with 2.50 mL of Biuret reagent.
3. The absorbance of
the samples was measured at 540 nm after 10 minutes.
4. Standard curve was
plotted.
5. The protein content
of the test sample was estimate using the standard curve.
(Note: All series
should include a zero protein (water) tube (reagent blank).
b) Lowry assay:
1. Obtain all 8 test
sample extracts from the other groups
2. 0.25 mL of each
protein (standard and test samples) was mixed with 2.5 mL of Lowry reagent 1.
3. The mixture was
incubated at room temperature for 10 minutes.
4. 0.25 mL of Lowry
reagent 2 was added and mix well immediately.
5. The mixture was
incubated at room temperature for 30 minutes.
6. The absorbance was
measured at 750 nm.
7. The standard curve
was plotted.
8. The protein content
of the test sample was estimated using the standard curve.
(Note: All series
should include a zero protein (water) tube (reagent blank)
RESULT
Protein Standard
|
Protein
Number
(Biuret)
|
B1
|
0.439
|
B2
|
0.509
|
B3
|
0.512
|
B4
|
0.542
|
B5
|
0.769
|
B6
|
0.995
|
|
Protein Number
(Lowry)
|
L1
|
0.132
|
L2
|
0.106
|
L3
|
0.406
|
L4
|
0.256
|
L5
|
0.115
|
L6
|
0.081
|
From this experiment, we used Biuret Assay and Lowry Assay to determine the protein content in the samples. In Biuret Assay, we used samples combine with the biuret reagent. Biuret reagent 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. Purpose of Biuret Assay is to prepare the standard curve, to determine the protein content in the sample and to analyse data from standard curve and unknown concentration of the samples.
To determine the protein concentration, we construct a standard curve after 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 was used to plot the graph of absorbance functioning as protein concentration called the standard curve for assay. This graph can be used to determine the other protein concentration in food samples.
From the graph, the highest protein concentration is braised fish, followed by fish, green bean, and the lowest is soy bean. The Lowry assay is based on the reaction of cupric ions with peptide bonds under alkaline conditions (the Biuret test). Protein samples are mixed with an alkaline solution containing copper sulphate (Cu2+ ions) which react with peptide bonds to produce Cu+ ions. As the amount of Mo(IV) and Mo(V) complex is dependent on the amount of Cu+ ions which is, itself, dependent on the amount of protein in the unknown sample, the colour produced is a direct reflection of protein concentration and, with the use of standards, can allow protein concentration to be determined.
Lowry Assay is one of the method that is used to determine the total level of protein in a solution. A standard curve is constructed to determine how much protein is represented by a particular absorbance reading. This is obtained by the Lowry reaction on a series of prepared solutions of gelatin at 0.1,0.2,0.3,0,4.0,5 and 0.6 mg/ml in water. The curve then is used to convert the absorbance reading for protein samples into protein concentration in the samples.
According to the graph that has been constructed, we able to find the protein concentration in the samples. From the graph, we found that the green bean (1.945 mg/ml) has the highest protein concentration followed by chicken (1.328 mg/ml), fish (1.320 mg/ml), peanut (1.219 mg/ml), and the lowest is dal beal (0.124 mg/ml)
CONCLUSION
In conclusion, both test can be used to determine the protein concentration. In biuret test, we obtain braised fish to have higher protein concentration while in Lowry test, green bean have the highest protein concentration.
QUESTIONS
1. Describe three alternative methods of determining protein concentration
UV
absorbance :
Protein concentrations can be determined
directly by ultraviolet spectroscopy because
of the presence of tyrosine and tryptophan
which absorb at 280 nm. Because the levels of these two amino acids vary
greatly from protein to protein, the UV absorbance per milligram protein is
highly variable.
BCA
Protein Assay :
The BCA Protein Assay is an alternative to
the Lowry assay. The key component in this assay is bicinchoninic acid (BCA)
that reacts with cuprous ions to generate an intense purple colour at 562 nm. Cuprous
ions are produced by the reduction of cupric ions by proteins in alkaline
solutions.
Dye-binding
method, (Bradford method, Bio-Rad protein assay):
In this assay, the 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. What is an “appropriate blank” and why?
Solution that will be a suitable blank on
the exact details of the method we are using.
So, use of blank ensures that all errors of
measurements that may be introduced into absorption spectrum from cuvette
material, solvent, temperature fluctuations, gases in the atmosphere are taken
into account.
REFERENCES
1. Ronald E. wrolstad, Terry E. Acree, Eric A. Decker, et al. (2005). Handbook of Food Analytical Chemistry: Water, Proteins, Enzymes, Lipids and Carbohydrates. John Wiley & Sons, Inc. Hoboken. New Jersey.
2. http://bio.classes.ucsc.edu/bio20L/MANUAL/Lab%203.pdf
3. https://socratic.org/questions/what-is-the-purpose-of-blank-solution-in-spectrophotometer
