Textbook - BIOLOGICAL CHEMISTRY - Gubsky Yu.I. - 2000

Chapter I. BIOMOLECULES AND CELLULAR STRUCTURES

CHAPTER 2. PROTEINS AND PEPTIDES

2.5. METHODS FOR ISOLATION AND ANALYSIS OF PROTEINS AND PEPTIDES

2.5.1. Methods of protein isolation and fractionation

The isolation of individual Proteins from Tissues, Cells, and biological fluids of living organisms (animals, plants, Bacteria, human Blood serum) is a common biochemical and biotechnological Procedure widely used to obtain medicinal agents (Hormones, Enzymes, interferons) or to study The properties of specific proteins in analytical biochemistry.

Class="center">Protein Isolation

To isolate proteins from biological objects, extraction using various Solvents is most commonly employed; the choice of solvent depends on the PHYSICOCHEMICAL PROPERTIES OF the protein or protein group to be obtained. When it is necessary to obtain proteins localized in specific subcellular Organelles and bound to biostructures (membranes, nuclear Chromatin, etc.), the protein isolation procedure includes:

- disruption of tissue and cellular structures (tissue homogenization, mechanical grinding, osmotic Shock);

- differential centrifugation of tissue homogenates to obtain isolated fractions of nuclei, Cell/35.html">Mitochondria, Endoplasmic reticulum membranes, Lysosomes, etc.;

- conversion of subcellular fraction proteins into a soluble state by treating biostructures with detergents or salt solutions;

- protein precipitation by salting out or using dehydrating Reagents such as ethanol (Cohn's method) or acetone.

Protein Fractionation

The result of these biochemical Procedures is typically The production of extracts containing a significant number of various proteins and non-protein components. Therefore, the next stage in isolating individual proteins is the fractionation of protein mixtures, which is carried out based on differences in the physicochemical Properties of Individual proteins (molecular weight, charge, solubility, chemical and biochemical activity). These same Methods also allow for the Determination of the respective physicochemical parameters of specific protein molecules.

Fractionation Methods:

1. Methods based on differences in the Molecular Weight of proteins:

- ultracentrifugation method (sedimentation analysis).

This method is based on The Use of high-speed ultracentrifuges, by means of which protein molecules (or other high-molecular-weight compounds) are subjected to centrifugal acceleration that exceeds the acceleration of gravity by hundreds of thousands of times (100,000-500,000 g). As a result of the significant centrifugal force, macromolecules settle (sediment) at a rate that depends on their size and molecular weight.

Determining the Sedimentation Rate of a protein molecule, expressed by the sedimentation coefficient s, makes it possible to calculate the molecular weight of the protein (M) using the Svedberg equation:

where R is the gas constant, T is the absolute Temperature, D is the protein diffusion coefficient, v is the partial specific volume of the protein, and p is the solvent density.

- Gel filtration method (Gel chromatography).

The method is based on differences in the rates of passage (filtration) of protein molecules differing in molecular weight through special gels that act as molecular sieves.

For gel filtration, polymeric compounds are used, most commonly derivatives of the polysaccharide dextran (commercial name — Sephadex), which have the form of granules with pores of a specific size. During passage through a chromatographic Column containing Sephadex granules, a mixture of proteins and other chemical compounds undergoes fractionation depending on molecular size and, accordingly, The ability to penetrate the gel network. The rate of passage of various molecules through molecular sieves is inversely proportional to their size and molecular weight.

2. Methods based on differences in the acid-base Properties of Proteins.

The amphoteric properties of proteins allow for their fractionation

by means of Ion-exchange chromatography and Electrophoresis.

The method of ion-exchange chromatography relies on the ability of charged protein molecules to selectively bind, via Ion Exchange, to specific sites on ion exchangers. Fractionation of components in protein mixtures is achieved by passing buffered protein solutions at various pH values through chromatographic columns packed with cation or anion exchangers.

For the chromatographic Separation of proteins via ion exchange, Cellulose-based ion exchangers are most commonly employed: specifically, the anion exchanger diethylaminoethyl cellulose (DEAE-cellulose) or the cation exchanger carboxymethyl cellulose (CM-cellulose).

3. Methods based on differences in the biochemical activity of individual proteins.

This group of methods is based on utilizing the varying affinity of natural proteins for specific chemical ligands, with which individual protein molecules actively interact in living organisms—Affinity Chromatography (derived from the English word “affinity”).

To perform affinity chromatography, a protein mixture is passed through a chromatographic column containing natural ligands specific to the protein targeted for isolation from a complex biological mixture. For instance, the isolation of enzymes relies on their specific binding to substrates, hormones to receptors, and IMMUNOGLOBULINS to their corresponding Antigens.