Proteins Practical Part 2تفاعلات الترسيب للبروتينات

Precipitation Reactions For Proteins

Proteins can be precipitated from solutions by a variety of positive and negative ions. Such precipitation is of importance in the isolation of protein in the deproteinization of blood and other biological fluids, and extracts for analysis and in the preparation of useful protein derivatives.

Precipitation by salts:

When an inorganic salt like ammonium sulphate or sodium sulphate is added to a solution of protein, the effective concentration of water available for the protein is decreased and the protein is precipitated, the process is known as salting out.

Procedure:

3 ml of protein solution + a pinch of solid ammonium sulphate with mixing until the solution is saturated →let this stand for 5 minutes + then add an equal volume of 40 % NaOH and 2 drops of 1% CuSO4 .(Biuret test) → blue colour .

Indicates that protein (albumin) is precipitated by full saturation.

 Precipitation by organic solvents:

Addition of organic solvent, such as acetone and alcohol decrease dielectric constant of solvent and displaces some water molecules associated with the protein and decrease the concentration of water in the solution. These effects tend to decrease solubility of protein.

Procedure:

1 ml of protein solution + 2 ml of ethanol , mix , and let it stand → a white precipitate is formed.

Precipitation by acidic agents:

Negatively charged ions like picric acid, trichloroacetic acid and sulphosalicylic acid are protein precipitants. They neutralize the positive charges on proteins and cause denaturation , resulting in precipitation of proteins.

Procedure:

1 ml of protein solution + 1 ml of picric acid solution → observe the formation of thick precipitate.

Repeat the test with trichloroacetic acid and salphosalicylic acid .

Precipitation by heavy metal ions:

When solution of  Lead acetate ,mercuric nitrate and other heavy metal salts are added to protein solution the cations interact with the negatively charged groups on the proteins causing. A precipitation as metal proteins and it may cause denaturation.

Procedure

1 ml of protein solution + 5 – 10 drops of basic lead acetate solution → observe the formation of precipitate

Repeat the test with mercuric nitrate solution.

Precipitation by heat and acid

Proteins are easily denaturated when subjected to heat treatment, because heating of proteins causes disorganization of peptide chains as a result of breaking of cross linkages with resultant unfolding of chains and marked changes in proteins. This called denaturation.

Procedure

10 ml of protein solution + over a flame →    a cloudy white ppt.

Add  a few drops of 1% of acetic acid .coagulation take place and albumin is precipitated