METHODS OF EXCTRACTION AND PURIFICATION OF PROTEINS

Some physical and chemical agents denature proteins. Denaturation is the loss of the secondary, tertiary and quaternary structure of a protein. This leads to a loss of some properties - solubility, biological activity.

Therefore special " sparing" methods are developed for the isolation of proteins.

Process begins with homogenization of biological material. This is grinding to the destruction of cells and subcellular structures. For this purpose we use pistillate or knife homogenizers, ball grinders, ultrasonic sound, method of tissue alternate freezing and thawing, method of " nitrogen bomb".

Then we make extraction of proteins by buffer mixtures with certain values of рН, organic solvents. The majority of proteins well dissolve in dilute solutions of salts.

For a fractionating and purification of proteins we use the following methods.

Salting-out is sedimentation of proteins from solution by adding solutions of salts alkaline and alkaline-earth metals. This method is used in clinical practice in the analysis of proteins of blood serum, for example, for division of globulins (drop out in a deposit at 50 % saturation of solution of ammonium sulphate) and albumins (in 100 % saturation).

Electrophoresis is a movement of charged dispersed particles in an electric field. Direction and speed depend on the charge and size of molecules. It is applied in clinical medicine in the analysis of proteins and peptide mixtures, blood serum.

Ultracentrifugation is a method of division of liquid disperses mediums to components under the influence of centrifugal force.

Chromatography (from Gr. chroma - colour) - a physical and chemical method of division and analysis/ Chromatography based on the distribution of mixture components between two phases - motionless (sorbent) and mobile (eluent).

We distinguish the following types of chromatography:

Adsorptive chromatography. Division of formulation constituents is based on their different sorbtion on solid adsorbent.

Distributive chromatography. The solid phase serves as support for a stationary fluid phase. Paper chromatography is a subtype of it.

Ion-exchange chromatography. We use suitable ion-exchange resin with the functional groups of which part of proteins interchange and stay on column while other proteins are unconstrained and eluate from it.

Gel chromatography, or a method of molecular sieves. It is based on the ability of small molecules to inpour into gel pores whereas the big molecules remain outside, moving together with a mobile phase downwards along a column. It allows dividing proteins with different molecular weight.

Perspective kinds of chromatography are High Performance Liquid Chromatography  (HPLC) and gas chromatography. The chromatography is one of the basic methods of biochemical researches. In clinical laboratories it applies to division and analysis of amino acids, proteins, carbohydrates, phospholipids, steroids in blood plasma, tissue extracts, urine.

FUNCTIONS OF PROTEINS

Catalytic function. The majority of enzymes known as biological catalists, are proteins. Now, some thousand enzymes are described.

Transport function. Respiratory function of blood, in particular oxygen transfer, is carried out by molecules of hemoglobin protein of erythrocytes. Albumins of blood serum take part in transport of lipids.

Protective function. In response to bacteria, toxins, viruses or alien proteins entering in organism protective proteins-antibodies are synthesized (immune defenses). A number of proteins of blood plasma are capable to coagulation that protects from wounds blood loss (physical protection).

Hormonal function. A series of hormones is presented by proteins or polypeptides, for example, pancreas hormone - insulin.

Structural function. In complex with lipids proteins participate in the formation of biomembranes of cells. Cytoskeleton structural proteins provide shape of cells and many organoids. Structural proteins are collagen in connective tissue, keratin in hair, nails, skin, elastin in a vascular wall, etc.

Nutrient (reserve) function. Proteins of egg (ovalbumins) are a source of nutrition for the fetus. The basic protein of milk (casein) also carries out nutrient function.

Receptor function. Protein receptors can be integrated into the cell membrane or in the cytoplasm. The receptor accepts a signal which is often a chemical substance.

Contractile (motor) function. Contractile function is inherent to muscular proteins (actin and myosin), to proteins of cytoskeleton and that provides discrepancy of chromosomes in the course of mitosis.

Other important functions of proteins - ability to sustain oncotic pressure in cells and blood, the buffer properties sustaining physiological рН value of internal environment, etc.

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