Purification of enzymes and other proteins usually start with precipitating them from solution. The stability of proteins in solution will depend mainly on the charge and hydration. Polar groups of the proteins (-NH2, COOH, OH groups) tend to attract water molecules around them to produce a shell of hydration. Any factor which neutralises the charge or removes water of hydration will therefore cause precipitation of proteins. The following procedures are used for protein precipitation:

1. Salting Out

When a neutral salt such as ammonium sulphate or sodium sulphate is added to protein solution, the shell of hydration is removed and the protein is precipitated. This is called salting out. As a general rule, higher the molecular weight of a protein, the salt required for precipitation is lesser. Thus globulins are precipitated with half saturation of ammonium sulphate; but albumin will need full saturation with ammonium sulphate for complete precipitation.

2. Iso-electric Precipitation

Proteins are least soluble at their iso-electric pH. Some proteins are precipitated immediately when adjusted to their iso-electric pH. The best example is Casein which forms a flocculent precipitate at pH 4.6 and redissolves in highly acidic or alkaline solutions. When milk is curdled, the casein forms the white curd, because lactic acid produced by the fermentation process lowers the pH to the iso-electric point of casein.

3. Precipitation by Organic Solvents

When an organic solvent is added to the protein solution, water molecules available for proteins are reduced, and precipitation occurs. Organic solvents reduce the dielectric constant of the medium which also favors protein precipitation. Hence, alcohol is a powerful protein precipitating agent. This may explain the disinfectant effect of alcohol.

4. Precipitation by Heavy Metal Ions

In alkaline medium, proteins have net negative charge, or are anions. To such a solution, if salts of heavy metals are added, positively charged metal ions can complex with protein molecules and metal proteinates are precipitated. Salts of Copper, Zinc, Lead, Cadmium and Mercury are toxic, because they tend to precipitate normal proteins of the gastro-intestinal wall. Based on this principle, raw egg is sometimes used as an antidote for mercury poisoning.

5. Precipitation by Alkaloidal Reagents

 Tungstic acid, Phosphotungstic acid, Trichloro acetic acid, Picric acid, Sulphosalicylic acid and Tannic acid are powerful protein precipitating agents. These acids lower the pH of medium, when proteins carry net positive charges. These protein cations are complexed with negatively charged ions to form protein-tungstate, protein-picrate, etc. and thick flocculent precipitate is formed. In clinical laboratory phospho-tungstic or trichloro acetic acid are usually used for precipitating proteins. Tanning in leather processing is based on the protein precipitating effect of tannic acid. Under certain conditions, proteins undergo denaturation, which is a mild form of precipitation reaction (Box 1). Heat coagulation is an irreversible precipitation process (Box 2 and Fig. 1).

Box1. Denaturation of Proteins

Box2. Significance of Heat Coagulation

Fig1. On heating, liquid white portion of egg becomes solid white coagulum

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مواضيع ذات صلة

Brown Adipose Tissue Promotes Thermogenesis

Hormones Regulate Fat Mobilization

Adipose Tissue is The Main Store of Triacylglycerol in The Body

Classification of Proteins

Study of Protein Structure

Imbalance in the Rate of Triacylglycerol Formation & Export Causes Fatty Liver

The Liver Plays A Central Role in Lipid Transport & Metabolism

HDL Takes Part in Both Lipoprotein Triacylglycerol & Cholesterol Metabolism

Tertiary Structure of Proteins

Secondary Structure of Proteins

Primary Structure of Proteins

Chylomicrons & Very-low Density Lipoproteins are Rapidly Catabolized