METHODS OF EXCTRACTION AND PURIFICATION OF PROTEINS

The SUBJECT Of BIOLOGICAL CHEMISTRY

Biochemistryis a science about chemical bases of processes of vital activity, studying chemical components of living cells, and also reactions and processes in which they participate. Its main task is the establishment of communication between a molecular structure and biological function of chemical components of living organisms.

The subject of medical biochemistryis the chemical processes occurring in a human body in norm and pathology, diagnostics and forecast on the basis of biochemical researches.

CHEMISTRY OF PROTEINS

Proteins are high-molecular nitrogen-containing organic compounds, which molecules are constructed of amino acids residues. Amino acid units linked together with peptide bonds.

Proteins compound the basis of both frames and functions of live organisms. Natural proteins are constructed of 20 various amino acids. These amino acids can be combined in the most different sequences, therefore they can form about millions various proteins. They provide existence of about thousand different living organisms, ranging from viruses to humans. Each organism is characterized by a unique number of proteins.

The content of proteins in various tissues of one organism is unequal. So, in a human body there are proteins in % from dry weight: in muscles - 80, in brain - 45, in bones - 20.

Element composition of proteins in terms of dry weight: C - 50-54 %; Н - 6,5-7,3 %; O - 21-23 %; N - 15-17 %; S - up to 0,5 %.

As a part some proteins contain in small amounts phosphorus, iron, manganese, magnesium, iodine, etc.

Theamount of nitrogen is rather constant in all proteins (about 16 %), therefore it is possible to define the quantity of protein in biological objects by the protein nitrogen.

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).

Electrophoresisis 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:

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

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

Ion-exchangechromatography. 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 chromatographyare 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.