L9. PROTEIN IDENTIFICATION

INTRODUCTION:

Biuret's test is a chemical test used for detecting the presence of peptide bonds. ( covalent bond formed between two peptides when the carboxyl group of the first one reacts with amino group of the second peptide). Polypeptides as proteins, are chains of amino acids link together by peptides bonds. 

A peptide bond can be broken by hydrolysis (the adding of water). In organims, protein molecules called enzymes facilitate the process.

The biuret reaction can be used to assess the concentration of proteins because peptide bonds accur with the same frequency per amino acid peptide. The intensity of the colour is directly proportional to the protein concentration. 

The solution to be tested in treated with a strong base followed by a few drops of copper (II) sulphate. If the solution turns purple, protein is present. Only peptides with a chain of at least 3 amino acids (two peptide bonds) give a significant measurable colour shift with these reagents.

Proteins in the alkaline environment reduce Cu2+ to Cu+, which forms a coordination complex with proteins, leading to a blue to pink-purple colour change. 

Our objectives:  

- Identify peptides bonds.

- Compare protein concentration in different foods.

MATERIALS:

- 7x250mL

- 6 test tubes

- test tube rack

- 6x10mL pipet

- mortar

- glass marking pen

- gloves

- goggles

- milk

- rice milk

- egg

- yogurt

- potato

- distilled water

- NaOH 20%

- 10 drops of CuSO4

PROCEDURE:

We are going to determine protein concentration in some foods: milk, rice milk, yogurt, potato and egg. Be careful, as we are going to test separately the egg white and the yolk. 

First of all we are going to dilute the protein.

1. Add 100mL of distilled water to each 250mL beaker. Label them with (M, RM, EW, EY, Y, P).

2. Separate the egg white and the yolk in another beaker.

3. Smash the potato in a mortar and add some amount of the smashed potato to the P beaker. 

Prepare the samples: 

1. Add 10mL of a dispersion of each food (M, RM, EW, EY, Y, P) to the indicate beaker. Calculate the final concentration. All the groups will use the same dispersion from the beakers.

2. Prepare 6 test tubes and label them. Add 2mL of each test tube of the every food dilution of each beaker.

3. Add 2mL of 20% NaOH dissolution.

4. Shake gently and add 5 drops of CuSO4 in each tube. Allow the mixture to stand for 5 minutes.

5. Note any colour change. Remember that proteins will turn solution pink or purple.

6. Compare the test tubes. 

RESULTS AND CONCLUSIONS:

Milk: positive
Rice milk: negative
Egg white: positive
Egg yolk: negative
Yogurt: positive
Potato: positive --> It had starch so it had to be negative. 
The positive result, are the foods of animals, because all they have proteins. Rice milk have starch like potato so they are negative. 
We ordered from the more concentration to the less concentration thanks to the colour. 
1. Egg white.
2. Potato and yogurt.
3. Milk.

QUESTIONS:

1.- Which food has protein?
Egg white, milk, yogurt, potato, ultimately, the animal food.

2.- Which food has more protein? why?
1 Egg white

2 Yogurt 

3 Cow milk => Because the animal food is rich in protein.

3.- Do you find any difference between rice and cow milk?
Rice milk doesn't have protein however cow milk does.

4.- Is there any difference among milk and yogurt? why?
Yes, yogurt has more protein than milk.