PRECIPITATION REACTIONS OF PROTEINS

 

PRECIPITATION REACTIONS OF PROTEINS

 

                Proteins have either a positive or a negative charge depending on the pH of the solution. If they are treated with either a metallic salt or strong acid, these charges are neutralised and the proteins precipitate out. Proteins form colloidal solution in which a shell or colour of water molecules surround the proteins molecule. If salt solutions of suitable concentration and charge are added, these ions destroy the shell of hydration, neutralise the charge on the protein molecule and the proteins precipitate out. Because of these properties proteins are preciitated out from their solution.

 

EXPERIMENT

Egg: white is the sources of protein used for the following experiments:-

 

I. Precipitation By Neutralisation and Dehydration:

 

Effect of concentration neutral salt solution:

 

(i) Half Saturation:

 

                To 3ml of protein solution add an equal volume of saturated ammonium sulphate solution, a white ppt is formed. This is precipitation of proteins by half saturation with ammonium sulphate.

 

(ii) Full Saturation:

                To 3ml of protein solution add solid ammonium sulphate with mixing untill the solution is saturated (i.e. there should be some undissolved salt in the bottom of the test tube). Let this stand for 5 minutes. Write your inference.

 

                Albumin being more hydrated than glubulin, is precipitated by full saturation while globulin is precipitated by half saturation. This property can be used for separating albumins from globulins. Higher the molecular weight of a protein, lower the concentration of salt required for precipitation from solution.

 

II. Precipitation By Heavy Metal Ions:

 

                When solutions of lead acetate, mercuric nitrate zinc sulphate & silver nitrate are added to protein solutions, the cations interact with negatively charged groups on proteins causing precipitation as metal-Proteinate and in many cases denaturation.

 

Note:- Raw eg: white is used as an immediate antidote in mercury poisoning and then an emetic to remove Hg4 ions which are held by albumin.

 

                To 1 ml of protein solution add 5-10 drops of lead acetate solution and observe the protein being precipitated. Repeat the experiment with solutions of 5% mercuric nitrate, zinc sulphate and silver nitrate solutions.


III. Precipitation by Alkaloidal Reagents:

                These alkaloidal reagents are used to get protein free filtrate in the clinical investigation.

 

                To 1ml of protein solution add few drops 20% sulph salicylic acid. The protein is precipitated. Repeat the experiment with 1% picric acid, 10% Trichloroacetic acid, phos photungstic acid and Esbach’s reagent (1gm of picric acid. 2gm of citric acid in 10m1 water). Esbach’s reagent is used for quantitative determination of albumin in urine.

 

IV. Precipitation by Strong Mineral Acid:

                This is used for detection of protein in urine.

 

Heller’s test

                To 3ml of strong HNO3 add drop by drop of the given protein carefully along the side of the tube. A white ring of precipitate due to the formation of acid meta-protein is observed.

 

V. Precipitation by Alcohol:

                To 1ml of protein solution add 2ml of ethanol, precipitation occurs, due to the denaturation of protein.

 

VI. Heat Coagulation Test:

                Take 3/4 test tube of protein. Hold the test tube in a slanting position and only boil the top layer, observe for coagulum. The lower half serves as a control. Add 1-3 drops of acetic acid, persistence of coagulum confirm the presence of protein.

 

                Denaturation is a change in the native character of the protein brought about by various chemical and physical agents. A denatured protein becomes less viscous, less soluble and looses its chemical property. All proteins do not coagulate on heating. Only albumin & globulin coagulate on heating.

 

                Precipitation reactions are useful in the separation of serum proteins Deproteinisation of blood or body fluids for chemical analysis is brought by precipitation reactions.

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