1. Biochemistry lab 4 (Proteins)
(BMS 233)
L. Noha Soliman

2. Physical , chemical properties
And
Identification tests Of
proteins

3. Introduction
Proteins are macromolecules composed of amino acids linked together through peptide bonds.
All amino acids found in proteins have this basic structure, differing only in the structure of the R-group or the side chain.

4. Physical Properties of Proteins

5. Physical properties of proteins
1- Proteins are colorless and tasteless.
2- Protein structures are of two distinct patterns - Globular proteins and fibrous proteins.
3- In general proteins have large molecular weights ranging between 5 X 103 and 1 X Due to the huge size, proteins exhibit many colloidal properties.

6. Physical properties of proteins
4- Proteins like the amino acids exhibit amphoteric property i.e., they can act as acids and alkalies.
5-The solubility of proteins depends upon the pH.
Lowest solubility is seen at isoelectric point.
The solubility increases with increase in acidity or alkalinity.
6-All the proteins show the plane of polarized light to the left, i.e., laevorotatory.

7. Solubility test Objective:
To test the solubility of amino acids and proteins in different solvent.
Principle:
Polar amino acids are more soluble in water than non-polar, due to presence of amino and carboxyl group which enables amino acids to accept and donate protons to aqueous solution.

8. Solubility test Materials:
Different amino acids and protein solutions.
Different solvents.
Test tubes
Water bath

9. Solubility test Method:
Place 0.5g of amino acid sample in 3 test tubes clean, dry containing 2ml of different solvents.
Shake the tubes thoroughly, then leave the solution for about one minute.
Record your result.

10. Solubility test
Results:
samples
Solvent

11. Chemical Properties of Proteins

12. Chemical properties of proteins
1- Hydrolysis of proteins: Proteins can be hydrolyzed by acid, alkali and proteases and broken down to peptides and mixture of amino acids. 2-Esterification: Proteins with reaction with alcohols gives its corresponding esters. 3-Amino acids reacts with amines to form amides. 4-When free amino acids or proteins are said to react with mineral acids like HCl, the acid salts are formed.

13. Detection of proteins

14. Precipitation by Organic Solvents
Principle:
Proteins in solution form hydrogen bonds with water.
Organic solvents like acetone, ether or ethanol when added to a protein solution in water, reduce the concentration of water molecules available for keeping the proteins in solution and thus decrease the number of hydrogen bonds.
The dielectric constant of the medium is also reduced causing aggregation, precipitation and denaturation of proteins.

15. Precipitation by Organic Solvents
Method:
1- Take 1 ml of protein solution in test tube.
2- Add 2 ml of ethanol.
3- Mix and let it stand for 5 minutes.
Observation:
A white precipitate is formed.
Inference:
Protein is precipitated by organic solvents.

16. Biuret Test Basis of test:
The nitrogen atoms in the peptide bonds (C-N bonds) in proteins bind with copper(II) ions Cu 2+ in Biuret reagent and produce a violet color

17. Biuret Test Method: Observations:
1- Add 2cm protein solution to a test-tube.
2- Add an equal volume of 5% potassium hydroxide (KOH) solution and mix.
3- Add 2 drops of 1% copper (II) sulphate CuSO4) solution and mix.
4- No heating is required.
Observations:
Violet color is formed.

18. Ninhydrin Test Principle:
Ninhydrin reacts with free α amino acids to give a bluish purple colored complex called Ruhemann’s purple.
Ninhydrin is a powerful oxidizing agent and causes oxidative decarboxylation of α amino acids producing an aldehyde.
The reduced Ninhydrin (hydrindantin) then reacts with ammonia and another molecule of Ninhydrin and produces bluish purple colored complex.
α -Amino acid + Ninhydrin Aldehyde + Hydrindantin CO2 + NH3
Hydrindantin + NH3 + Ninhydrin Blue complex
Ninhydrin reagent: 0.2% of Ninhydrin in acetone.

19. Ninhydrin Test Method 1-Take 2 ml of protein solution in a test tube.
2- Add 10 drops of Ninhydrin.
3- Heat to boiling
Observation
Bluish purple color is formed.
Inference
Indicates the presence of free α-amino acids..

20. Xanthoproteic Test Principle:
The benzene ring system in tyrosine and tryptophan undergo nitration on treatment with strong nitric acid at elevated temperature forming a yellow precipitate.
The yellow precipitate turns orange due to ionization, in alkaline medium.

21. Xanthoproteic Test Method
1- Take 2 ml of protein solution in a test tube.
2-Add 1ml of conc. Nitric acid and mix.
3- Heat the solution for one minute and cool it under tap water.
4- Divide the contents into two parts.
5- To one part of the solution, add 2 ml of 40% sodium hydroxide.
Observation
In acid medium yellow color is formed.
In alkaline medium orange color is formed.
Inference
 Indicates the presence of aromatic amino acids. (Tyrosine and tryptophan).