Salt Saturation Test

1. Salt Saturation Test

2. Background • Proteins which are collide in nature are kept in solution by two factors • Electric charge: A large no of electric charge are present on the surface of protein molecules. The similarly charged particles repel each other so this prevents coalescence of particles • Shell of hydration: each molecule is surrounded by film of water known as shell of hydration this also prevents coalescence of particles

3. Principle • When an inorganic salt like ammonium sulphate is added to protein solution, the effective concentration of water available for the protein is decreased and protein is precipitated. The amount of ammonium sulphate required to precipitate a collide depends upon the surface area of the particles. Thus small molecule like albumin having relatively large surface area are precipitated only by full saturation with ammonium sulphate. The large casein and gelatin molecules have a smaller surface area, therefore both are precipitated by half saturation with ammonium sulphate.

4. Principle • Albumin due to large surface area holds much more water than globulin . Hence albumin requires much higher concentration of the salt than globulin to be precipitated. This property used for separation of albumin from globulin. The general rule is higher the molecular weight of protein lesser is the surface area and lesser is hydration and lower is the concentration of salt required for the precipitation.

5. Reagents • Solid ammonium sulphate • Saturated solution of ammonium sulphate • 40% NaOH • 1% CuSO4

6. Half Slat Saturation Test Procedure: • Take 5 ml of given solution and 5 ml of saturated solution of ammonium sulphate in a test tube shake vigorously and allow to stand for 5 minutes (because the volumes of given solution and saturated ammonium sulphate solution are equal so now the saturated ammonium sulphate solution become half saturated) • Filter and perform Biuret test with the filtrate using an equal volume of 40% NaOH and 2-3 drops of CuSO4

7. Half Slat Saturation Test Interpretation: • Biuret test is positive i.e. violet color appears in case of albumin and negative i.e. no change in case of globulin.

8. Full Slat Saturation Test Procedure: • Take 5 ml of given solution and add solid ammonium sulphate in a test tube while mixing until the solution is saturated i.e. there should be some undissolved salt at the bottom. shake vigorously and allow to stand for 5 minutes • Filter and perform Biuret test with the filtrate using an equal volume of 40% NaOH and 2-3 drops of CuSO4

9. Full Slat Saturation Test Interpretation: • Biuret test is negative i.e. no change

10. Precipitation of protein by concentrated salt solution