PROJECT 5 : PROTEIN

EXPERIMENT 5: PROTIEN (AMENDED)

Introduction:

Proteins are large biomolecules that consisting of one or more long chains of amino acid residues. Proteins are importans for catalysing metabolic reactions, DNA replication, responding to stimuli and also transporting molecules. 

Protein concentration can be determine by using two methods which is using Biuret assay method and also Lowry assay method. The advantage of using Biuret assay is method include that only few materials  interfere with it. it also can be done in a short time and does not depend on the amino acid composition of the protein. However the disadvantages of this method is low sensitivity and that it requires at least 1 mg of protein. The advantage of Lowry assay method is sensitive to low concentrations of protein. The major disadvantage of the Lowry method is the narrow pH range within which it is accurate.

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 0.6mg/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

            c.       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.

Result:

 

Discussion:

There are many methods for determining protein concentration. Some of these methods depend on the reaction of reagents with peptide bonds or amino acid side chains of the protein, others depend on the binding of a reagent (dye) to the protein. In chromogenic methods, the absorbance of coloured product formed by the protein and an organic molecule is measured. Protein concentration can also be ddetermined from the protein’s own (intrinsic) UV absorbance. However, these methods may give different results for different proteins of the same concentration. Beside that, different methods can yield somewhat different results for the same protein.

In this experiment, Biuret test was used to determine concentration of protein. 0.50 ml of each protein (standard and test samples) was mixed with 2.50 ml of biuret reagent. Then, the absorbance of the sample was measured at 540 nm after 10 minutes. Molecule 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. The determination of protein concentration is done using a calibration curve created using samples of known concentration. The benefits using Biuret test to determine protein concentration are simple procedure and easy to handle in laboratory. Besides that, the chromogenic rate is constant for most proteins. However, sample with a low protein concentration cannot be measured. This due to reaction of chromogenic was influenced by high concentration of trisaminomethane, amino acids, and ammonium ion.

Based on the graph that has been constructed, boiled fish recorded the highest protein concentration which is 9.25. While, the protein concentration of fish that was observed is 7.9 which is lower than concentration of protein of boiled fish. Soybean recorded the lowest protein concentration which is 1.30, while peanut protein concentration was 1.65. While, the protein concentration of beef, chicken, red bean and dal bean was 1.8, 2.50, 3.60 and 4.75 respectively. 

Lowry assay is a biochemical assay for determining the total level of protein in a solution. The protein sample in animal and plant sources such as beef, chicken, fish, soybean, peanut, red bean, dal bean, and green bean is tested using Lowry test. From the experiment, chicken has the highest protein content which is 0.50 mg/mL as compared to beef and fish which is 0.41 and 0.49 respectively. Boiled fish had lower protein content than fresh fish, which is 0.27 mg/mL. This is due to the denatured of protein during heating process.

           Among plant sources, green bean has the highest protein content, 0.65 mg/mL. This is followed by peanut, 0.47mg/mL. Red bean has 0.40mg/mL of protein while soybean and dal bean has 0.26mg/mL and 0.22 mg/mL respectively.     

 Question:

1. Describe three alternative methods of determining protein concentration.

Dye-binding method. Dye Coomassie Blue G-250 is dissolved in an acidic solution causing it to absorb at 465 nm which is reddish brown. When the dye that have negatively charged binds to the positively charged protein molecule the absorbance undergoes a shift to 595 nm which is blue colour. This shift in absorption maximum is proportional to protein concentration over a broad range.

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.

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. This method also include high sensitivity so valuable protein samples can be recovered
2. What is an “appropriate blank” and why? 

Appropriate blank is a reference measurement of the transmitted light as a function of wavelengths is stored in memory. An appropriate blank is control samples, blanks, standards, dummy analyses, and system suitability checks to increase confidence in measurements. Blank is just a solution that does not contain the thing you are measuring, and this gives a base, or zero reading. This helps to ensure the reading is correct. 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:

As a conclusion from the experiment that we conduct, we determined that in Lowry test, green beans shows the highest protein number that is 1.945 when compared to the other sources of protein. While for the Biuret test, braise fish has the highest protein number which is 1.138.

Reflection : 

LENDRA NALAT ( D20141067021)

In this experiment, I have learned how to determine concentration of protein using Biuret test at 540 nm and Lowry test at 750 nm. The concentration of protein of unknown protein sample can be determined based on the graph that has been constructed. Biuret test can be used to determine the concentration of protein because peptide bonds occur with the 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. Besides that, I also learned to always be careful when doing experiment and follow the instruction correctly. An error may occur if the experiment do not doing properly.
 
TING MEE PING (D20141067055)

From this experiment, I have learnt that protein can be extract from animal and plant sources. Protein content in food samples can be determine by Biuret assay or Lowry assay. From the experiment, protein from plant sources also can be high as animal sources. Heating can cause protein content to reduce as protein denature in high temperature.  

CHRIS ROXA ANGELLA SUPAIN (D20141067094) 

From this experiment, I had learn the method that can be used to determine the protein concentration in the food. Beside that I had learn that Lowry assay is more accurate than Biuret assay since it is quite sensitive. I also learn how to use spectrophotometer to detect the absorbance of food samples. This is the last experiment that we conducted and overall I had enjoyed it and gain a lot of knowledge. I would like to thank our instructor, Miss Atiekah for helping me during this semester.

Reference:

Zhou, P., & Regenstein, J. M. (2006). Determination of total protein content in gelatin solutions with the Lowry or Biuret assay. Journal of food science, 71(8), C474-C479.

N. A. Khan and K. N. Singh. 2014. Laboratory manual of biochemistry. New Delhi: Daya Pub. House