LAB 4 : PROTEIN ANALYSIS

LAB 4

PROTEIN ANALYSIS

INTRODUCTION

Proteins are polymers of amino acids. Twenty different types of amino acids occur naturally in proteins. Proteins differ from each other according to the type, number and sequence of amino acids that make up the polypeptide backbone. As a result they have different molecular structures, nutritional attributes and physiochemical properties. Proteins are important constituents of foods for a number of different reasons. They are a major source of energy, as well as containing essential amino-acids, such as lysine, tryptophan, methionine, leucine, isoleucine and valine, which are essential to human health, but which the body cannot synthesize. Proteins are also the major structural components of many natural foods, often determining their overall texture, e.g., tenderness of meat or fish products. Isolated proteins are often used in foods as ingredients because of their unique functional properties, i.e., their ability to provide desirable appearance, texture or stability. Typically, proteins are used as gelling agents, emulsifiers, foaming agents and thickeners. Many food proteins are enzymes which are capable of enhancing the rate of certain biochemical reactions. These reactions can have either a favorable or detrimental effect on the overall properties of foods. Food analysts are interested in knowing the total concentration, type, molecular structure and functional properties of the proteins in foods.

OBJECTIVES

• Learn the principles of protein assays.
• Determine protein concentrations using the Biuret Protein Assay.

MATERIALS

Stock solution of Bovine Serum Albumin (BSA) : 10mg/ml

Deionized water (dH2O)

Test tubes and stand

Pipette

Biuret reagent

Spectrophotometer

Protein samples

METHODOLOGY

(A) Preparation of Biuret Reagent

Added 300ml of 10 % (w/v) NaOH to 500ml of a solution contained 0.3% copper sulfate pentahydrate and 1.2% sodium potassium tartarate, with stirred. Then, diluted to 1 liter.

(B) Protein Preparation

1. 2 sets of test tubes from number 1 to 6 were prepared and bovine serum albumin (BSA) stock solution (10mg/ml) were prepared according to the concentration listed below :

Tube	BSA conc. (mg/ml)	H2O	BSA stock (ml)
1	0	1.0	0
2	1	0.9	0.1
3	2	0.8	0.1
4	3	0.7	0.3
5	4	0.6	0.4
6	5	0.5	0.5
7	6	0.4	0.6

2. The prepared test tubes for protein samples were duplicated and 1ml of the protein samples were pipette carefully into each tubes.

3. 2ml of biuret reagent were added to every tube : 14 tubes were needded for the standard curve and duplicated tubes for protein samples.

4. The tubes was covered with parafilm and briefly vortex to ensure the protein standards/samples with the biuret reagent were thoroughly mixed.

5. Stand the tubes for 15 minutes.

6. Spectrophotometer were switch on and adjust the wavelength to 550nm.

(C) Determine Protein Concentration

1. 1ml of solution from the tubes were transfered into a cuvette and wipe the cuvette gently with a paper towel to remove fingerprints and dust.

2. Set the absorbance to 'zero'. It will served as 'blank'.

3. Absorbance of the other standards and sample protein will be measured using step in (1).

* DO NOT blank the instrument again.

4. Absorbance of the each standards and samples were recorded.

5. Graph of standard curve using the the absorbance value of protein standards will be plotted and absorbance values of the protein samples were interpolated.

RESULT

Test Tubes	BSA 1	BSA 2	Average BSA
0	0.120	0.142	0.1310
1	0.299	0.334	0.3165
2	0.352	0.368	0.3600
3	0.461	0.428	0.4445
4	0.494	0.521	0.5075
5	0.562	0.559	0.5605
6	0.600	0.592	0.5960

               

Protein Concentration	Absorbance Value
Soy	0.490
Albumin	0.411

The ratio of soy = 1 : 20

                           = 4.7 mg/ms

The ratio of albumin = 1 : 10

                                  = 3.6 mg/ms

DISCUSSION :

In this experiment, we have to investigate about the protein contents (concentration) in the soy and albumin.  We used Biuret reagent test to test the content of protein in those samples. 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-colour coordination complexes in an alkaline solution. 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 colour and hence the absorption at 540 nm is directly proportional to the protein concentration according to the Beer-Lambert law.

Before we tested the sample, we have plot the standard curve. After that, we have to dilute the samples because if we do not dilute those sample, we cannot get the amount of the protein content. From our results, we found out that soy sample have the highest content of protein compare to albumin sample. The amount of protein contents in soy sample is 4.7 mg/ms while the albumin sample is 3.6 mg/ms.

CONCLUSION :

Biuret test is used to test for the presence of peptide bond. The Biuret reagent contains copper ions which give it a blue color. The copper ions will interact with a compound that contains two or more peptide bonds, resulting in the formation of a violet/purple-colored product. When a compound does not have at least two peptide bonds, it will not react with the Biuret reagent, and no purple color will appear (solution will remain a shade of blue due to the copper ions). Positive result for Biuret test will show a purple colour solution, changes from blue. Among starch solution, glucose solution, protein and water, peptide bond only exists in protein. Therefore, only protein will show colour changes from blue to purple. All samples of protein with more than two peptide bonds will show colour changes from blue to purple.

REFERENCES :

Biuret test. Retrieved on April 1, 2013 from http://en.wikipedia.org/wiki/Biuret_test

Lisa Porter (2011). Egg protein levels. Retrieved on April 1, 2013 from http://www.livestrong.com/article/470871-egg-protein-levels/#ixzz2PH9KhV7I

Cavemangreg (2011). All about eggs. Retrieved on April 1, 2013 from http://www.paleodietandliving.com/paleo-diet/all-about-eggs/