Lab Report 4: Protein

INTRODUCTION
In the experiment to determine the protein concentration, Biuret assay and Lowry assay are used. The biuret test is a chemical test used to detect the presence of peptide bonds. In the presence of peptides, a copper(II) ion forms violet-coloured coordination complexes in an alkaline solution. The Biuret reaction can be used to assess the concentration of proteins because peptide bonds occur with same frequency per amino acid in the peptide. The intensity of the colour and hence the absorption at 540 nm, is directly proportional to the protein concentration, according to the Beer-Lambert law. Meanwhile, 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 colour change of the sample solution in proportion to protein concentration, which can then be measured using  colourimetric techniques.

MATERIALS

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














Protein Number for Samples

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

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