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Isolation and purification of proteins is carried out in stages.

1. Homogenization- this is a thorough grinding of objects of biochemical research to a homogeneous, that is, a homogeneous state, that is, proteins undergo thorough disintegration up to the destruction of the cell wall.
In doing so, they use:
A) Warring-type knife homogenizers;
b) pestle homogenizers Potter - Elveheim;
V) ball and roller mills - for denser objects;
G) the method of alternate freezing and thawing, while the rupture of the cell wall occurs under the action of ice crystals;
e) the method of "nitrogen bomb" - under high pressure, the cells are saturated with nitrogen, then the pressure is abruptly released, gaseous nitrogen is released, which, as it were, explodes the cell from the inside;
e) Ultrasound, various press methods, digestion of cell walls with enzymes. In most cases, heat is released during homogenization, while many proteins can be inactivated, so all procedures are carried out in cold rooms at t 0 or the raw materials are cooled with ice. At the same time, the volume and time of cell destruction, as well as the operating pressure, are carefully controlled. An ideal homogenizate is one that can be further extracted.

2. Protein extraction, that is, their transfer to a dissolved state; most often, extraction is carried out together with grinding at the same time.

Extraction is carried out:
A) dissolution in 8-10% salt solutions;
b) using buffer solutions with pH from acidic to slightly alkaline (borate, phosphate, citrate, tris - buffer: a mixture of trisaminomethane with NH2 - CH3 + HCl;
V) precipitation of proteins with organic solvents (ethanol, methanol, butanol, acetone and their combinations), while splitting the protein-lipid and protein-protein components, that is, the destruction of HSB.

3. Purification and fractionation of proteins. After extraction, the mixture is separated or fractionated into individual proteins and further purified:

a) salting out- this is the process of precipitation of proteins by neutral salt solutions of alkaline and alkaline earth metals.

salting out mechanism– added anions and cations destroy the hydrated protein shell of proteins, which is one of the stability factors of protein solutions. Most often, solutions of Na and ammonium sulfates are used. Many proteins differ in the size of the hydration shell and the magnitude of the charge. Each protein has its own salting out zone. After removal of the salting out agent, the protein retains its biological activity and physicochemical properties. In clinical practice, the salting out method is used to separate globulins (with the addition of 50% ammonium sulfate solution (NH 4) 2SO 4 a precipitate forms) and albumins (with the addition of 100% ammonium sulfate solution (NH 4) 2SO 4 a precipitate forms).

Salting out is influenced by:
1) nature and concentration of salt;
2) pH environments;
3) temperature.

The main role is played by the valencies of the ions. Therefore, the effect of salt is evaluated by the ionic strength of the solution μ:

, that is, the ionic strength of the solution (μ) is equal to the product of ½ of the concentration of each ion (C) and the square of its valence (V).

The Kohn method is a type of salting out. At the same time, extraction and precipitation of the components take place. By successively changing the temperature (usually low t o -0 + 8 o C), the pH of the solution and concentrated ethanol, up to 18 protein fractions are sequentially isolated from the blood plasma.

Kohn Method used in pharmaceutical production in the production of blood substitutes;
b) chromatography methods. The Russian scientist Mikhail Tsvet (1903) is considered the founder of the development of chromatographic methods of analysis. Currently, there are many varieties of it. The method is based on the ability of substances to be specifically adsorbed on an adsorbent enclosed in a column or placed on some carrier. When this occurs, the separation of the analyzed substances and their concentration in a strictly defined adsorbent layer. Then, appropriate eluents (solvents) are passed through the column, which weaken the adsorption forces and wash out the adsorbed substances from the column. Substances are collected in the fraction collector.

Fundamental to chromatography is distribution ratio, which is equal to the ratio of the concentration of a substance in mobile phase to the concentration of the substance in stationary phase(or stationary phase).

Stationary stationary phase– may be solid or liquid or a mixture of solid and liquid.

mobile phase- liquid or gaseous, it flows through the stationary, or is passed through it.

Depending on the type of stationary and mobile phase, there are various modifications of chromatographic analysis.

Adsorption- is based on the different degree of adsorption of proteins by the adsorbent and their solubility in the corresponding solvent.

Adsorbents used are silicic acid, Al 2 O 3 , CaCO 3 , MgO, charcoal. The adsorbent in the form of a suspension with a solvent (usually with a buffer solution) is packed in a column (glass vertical tube). The sample is applied to the column, then a solvent or mixture of solvents is passed through it.

The separation is based on the fact that substances with a higher K distribution. (B) moving along the column at a faster rate. Fractions are collected using a fraction collector.

Partition chromatography- based on the distribution of a mixture of proteins between two liquid phases. Separation can take place on special chromatographic paper, as well as in columns, as in adsorption. The solid phase in this case serves only as a support for the liquid stationary phase. Chromatographic paper has the property of retaining water between its cellulose fibers. This water is a stationary stationary phase. When a non-aqueous solvent (mobile phase) moves through the paper under the action of capillary forces, the molecules of the substance deposited on the paper are distributed between the two phases in accordance with their distribution coefficient. The higher the solubility of a substance in the mobile phase, the further it will move along the paper along with the solvent.
In the case of distribution of chromatography on a column, the carriers are cellulose, starch, silica gel, etc., the stationary phase is water. When applied to the column, the substances of the mixture move along the column at different speeds, taking into account Krasp.

Rf for each compound under standard conditions is a constant value.
Ion exchange chromatography - based on the attraction of oppositely charged particles. For this, various ion-exchange resins are used: cation-exchange resins contain negatively charged groups - sulfonated styrenes and CMC, which attract positively charged ions of the substances under study. They are also called acid ion exchangers.
Anion exchange resins, or basic ion exchangers, contain positively charged groups that attract negatively charged protein molecules.
Trimethylaminostyrene is a derivative of styrenes and cellulose.
Depending on the q of the proteins to be separated, appropriate ion exchangers are used, with which certain proteins interact, while others freely leave the column. Proteins "precipitated" on the column are removed using more concentrated saline solutions or by changing the pH of the eluent.
Affinity chromatography (or affinity chromatography) is based on the principle of selective interaction of proteins or other macromolecules with specific substances immobilized on carriers - ligands (this can be a coenzyme if an enzyme, antibody, antigen, etc. is isolated. Due to the high specificity of proteins, immobilized ligands are attached to it only one protein per mixture Washed out with buffer mixtures with altered pH or altered ionic strength.
The advantage is the ability to isolate a given substance of a high degree of purity in one step.
The gel filtration method or molecular sieve method is a type of permeation chromatography.
The separation of molecules according to size and shape is based on the properties of the molecular sieve that many porous materials have, such as organic polymers with a three-dimensional network structure that gives them the properties of gels. Gel filtration is the separation of substances using gels based on differences in the size of molecules (sepharose, sephadex, sephacryl, biogels, etc.). Under the action of epichlorohydrin, the polysaccharide chains of dextran (synthesized by microorganisms) are crosslinked into a network structure, become insoluble in water, but retain a high affinity for it. Due to this hydrophilicity, the resulting grains (called Sephadex) swell strongly to form a gel, which is filled into the column. The method is based on the fact that large molecules do not penetrate into the internal aqueous phase, and smaller molecules first penetrate into the pores of the "sieve", as if stuck in them, and therefore move at a lower speed. Accordingly, proteins with a higher Mr are the first to enter the receiver. Recently, porous glass beads have been increasingly used as a molecular sieve in permeation chromatography.
The electrophoretic method in biochemistry is based on the difference in the speed of movement of molecules in an electric field (amino acids, peptides, proteins, nucleic acids).
The speed difference depends on:
1. on the q of the molecule: the greater the mobility of the molecules, the greater the total q. The q value depends on pH;
2. on the size of the molecules: the larger the molecules, the less their mobility. This is due to the increase in friction forces and electrostatic interactions of large molecules with the environment;
3. on the shape of molecules: molecules of the same size, but different shapes, for example, fibrils and protein globules have different speeds. This is due to differences in the forces of friction and electrostatic interaction.
Types of electrophoresis
a) Isoelectric focusing. Separation occurs on a vertical column in deg. both pH and voltage. With the help of special carriers of ampholytes, hail is established in the column. pH from 0 to 14. A mixture of substances is placed in the column, and an electric current is connected. Each of the components moves to that part of the column where the pH value corresponds to its isoelectric point and stops there, that is, it is focused.
Advantage: separates, purifies and identifies proteins in one step. The method has a high resolution (0.02 pI).
b) Isotachophoresis is electrophoresis on supporting media. After turning on the electric current, the ions with the highest mobility move to the corresponding electrode first, with the lowest - the last, having intermediate mobility - are located in the middle.
c) Disc electrophoresis - the device consists of two vessels with a buffer - upper and lower, connected by vertical tubes containing a gel of different pores. As ionized particles move under the action of an electric current. Higher porosity is at the top of the gel.
d) Immunoelectrophoresis - a method combining electrophoresis with immunodiffusion (for the detection of antigens in complex physiological mixtures). A mixture of antigens and a mixture of antibodies are placed perpendicular to each other on a special carrier. When the electric current is turned on, they are separated into individual substances and diffuse on the gel carrier. At the meeting point of the antigen with the corresponding antibody, a specific precipitation reaction occurs in the form of an arc. The number of formed arcs corresponds to the number of antigens.

Methods for determining Mr proteins

At a large number proteins, the chemical composition and sequence of amino acids has not been established (1010–1012 proteins), therefore, Mr. Various methods are used for this.
a) Sedimentation method - the determination of Mr is carried out in special centrifuges (the first centrifuge was proposed by the Swedish biochemist Svedberg), in which it is possible to create a centrifugal acceleration that is more than 200 thousand or more times the acceleration of the earth's gravity. Mr is determined from the V sedimentation of the molecules. As molecules move from the center to the periphery, sharp border solvent protein. The sedimentation rate is expressed in terms of the sedimentation constant (S):

where V is the speed of movement of the protein-solvent interface (cm/s);
 is the angular velocity of the rotor (rad/s);
 is the distance from the center of the rotor to the middle of the cell with the protein solution (cm).
The value of the sedimentation constant S, which is equal to 110–13 C, is conditionally taken as 1 and is called 1 Svedberg (S). S for proteins ranges from 1-50 S, sometimes up to 100 S.
Mr of proteins is determined by the Svedberg equation:

where R is the universal gas constant;
T - absolute temperature by Kelvin;
S is the sedimentation constant;
D is the diffusion coefficient;
 is the density of the solvent;
V is the partial specific volume of gas.
This method is expensive due to the use of equipment.
More simple and cheaper:
b) Gel filtration in a thin layer of Sephadex.
The protein path length (in mm) is logarithmic to Mr.
X - Mr of the desired protein on the calibration graph.
c) Disk electrophoresis in a polyacrylamide layer - there is also a relationship between the logarithm Mr of the calibration proteins and their path length.

Methods for determining the homogeneity of proteins

The degree of purity of the isolated protein is determined by:

• ultracentrifugation;
• disk-electrophoresis method;
• various immunochemical methods;
• determination of protein solubility (the Northrop method) is based on the phase rule, according to which the solubility of a pure substance under given experimental conditions depends only on temperature, but does not depend on the concentration of the substance in the solid phase.

If the protein is homogeneous, then one inflection is obtained on the graph (a), if there are protein impurities (b, c), then we will get several inflections of the saturation curve. All proteins have their own individual solubility curves.

480 rub. | 150 UAH | $7.5 ", MOUSEOFF, FGCOLOR, "#FFFFCC",BGCOLOR, "#393939");" onMouseOut="return nd();"> Thesis - 480 rubles, shipping 10 minutes 24 hours a day, seven days a week and holidays

Kaisheva Anna Leonidovna Mass-spectrometric identification of proteins and protein complexes on the chips of an atomic force microscope: dissertation... candidate of biological sciences: 03.01.04 / Kaisheva Anna Leonidovna; [Place of protection: Nauch.-issled. in-t biomed. chemistry them. V.N. Orekhovich RAMS].- Moscow, 2010.- 104 p.: ill. RSL OD, 61 10-3/1308

Introduction

Chapter 1 Literature Review 10

1.1. Analysis of scientific and technical groundwork in the field of highly sensitive proteomic technologies

1.2 Characterization of hepatitis C virus 20

1.2.1 Methods for diagnosing hepatitis C 22

1.2.2 Serological protein markers of hepatitis C 25

Chapter 2. Materials and methods 28

2.1 ACM chips 28

2.2 Protein preparations and reagents 29

2.3 AFM analysis 30

2.4 Sample preparation for mass spectrometric analysis 31

2.5 Mass spectrometric analysis 33

2.5.1 MALDI-MS analysis of proteins on the surface of the AFM chip 33

2.5.2 ESI-MS analysis of proteins on the surface of the AFM chip 34

Chapter 3 Results and Discussion 35

3.1 MS - identification of proteins caught by "chemical fishing" on the surface of the AFM chip from the analyte solution

3.2 MS identification of proteins biospecifically captured on the surface of an AFM chip from an analyte solution

3.3 MS identification of proteins on the surface of an AFM chip biospecifically isolated from blood serum samples

Conclusion 83

Literature

Introduction to work

The relevance of the work.

One of the priority areas in modern biochemistry is the creation of effective analytical methods for proteomic analysis, the main task of which is to detect and inventory proteins in the body, study their structure and functions, and identify protein interactions. The solution of this problem will allow creating new systems for diagnosing diseases and their treatment. Standard methods of modern proteomic analysis are based on the separation of multicomponent protein mixtures using chromatography, electrophoresis in combination with mass spectrometric methods (MS) for protein identification. Despite the undoubted advantage of standard MS analysis in terms of speed and reliability of identification of protein molecules, it has significant application limitations due to low

concentration sensitivity of the analysis at the level of 10" "10" M and a high dynamic range of protein content in biological material. At the same time, the vast majority of functional proteins, including biomarkers of such socially significant diseases as viral hepatitis B and C, tumor markers, etc. ., are present in the blood plasma at concentrations of 10" Mi less.

One of the ways to overcome this methodological limitation of the concentration sensitivity of analysis is to use biomolecular detectors, which allow the detection of single molecules and their complexes and theoretically have no concentration sensitivity limitations. Biomolecular detectors include detectors based on nanotechnological devices such as atomic force microscopes (AFM), nanowire detectors, nanopores, and a number of other detectors. The unique sensitivity of AFM detectors makes it possible to visualize individual protein molecules and count their number. When using AFM as a biomolecular detector, it is necessary to use special chips that allow the concentration of biological analyte macromolecules from a large volume of incubation solution on a limited chip surface. The studied protein objects can be concentrated on the chip surface both due to physical or chemical adsorption, and due to biospecific interactions (AFM-biospecific fishing).

However, in practice, the limitation of the use of AFM-based nanodetectors is that, despite the possibility of visualizing individual protein molecules on the chip surface, such detectors are not able to identify them, which is especially important in the study of complex protein mixtures, including biological material. Therefore, the development of an analysis method that complements the capabilities of the AFM method seems to be an urgent task. To date, the only proteomic method that allows unambiguous and reliable identification of protein molecules is MS analysis. In the dissertation work, an approach was developed that combines the high sensitivity of the AFM method and reliable MS identification for the detection of proteins and their complexes from an analyte solution.

Purpose and objectives of the study.

The purpose of this work was the mass spectrometric identification of proteins and protein complexes detected in the biomaterial using atomic force microscopy.

To achieve this goal, the following tasks were solved:

A scheme for the MS identification of proteins caught on the surface of an AFM chip using chemical or biospecific fishing has been developed;

Conditions for enzymatic hydrolysis of proteins on the surface of an AFM chip for subsequent MS identification have been developed;

MS-identification of model proteins on the surface of the AFM chip was carried out;

MS-identification of proteins on the surface of the AFM chip biospecifically isolated from a multicomponent mixture (serum) was carried out.

Scientific novelty of the work .

In the dissertation, a scheme was developed that allows MS identification of proteins and protein complexes caught from a solution or a multicomponent mixture on the surface of an AFM chip. For this, the optimal conditions for sample preparation were selected, including the mode of hydrolysis (temperature, humidity, composition of the trypsinolytic mixture, trypsinolysis time) of protein molecules covalently and non-covalently immobilized on the surface of the AFM chip. The peculiarity of this work was that, compared with the standard proteomic protocols of enzymatic hydrolysis, the preparation of samples for MS analysis was carried out not in solution, but on a limited area.

chip surface. The developed scheme made it possible to effectively carry out MS analysis and identify both individual proteins and protein complexes on the surface of the AFM chip. MS analysis of proteotypic peptides of the studied proteins was carried out using two types of ionization (MALDI And EST) and two types of detectors (TOF and ion trap). The developed scheme for coupling AFM-biospecific fishing and MS was also successfully tested for the detection of protein markers of viral hepatitis C (HCV) (HCVcoreAg and E2) in blood serum samples.

The practical significance of the work .

The results of this work make it possible to create highly sensitive proteomic methods without the use of labels and additional sample preparation procedures for the detection of proteins found in low concentrations in biological material, including in blood serum. An approach based on atomic force microscopy and mass spectrometry has been proposed, which will make it possible to detect and identify protein markers of hepatitis C virus in human blood serum.

The approach can be used in developments aimed at creating new diagnostic chips, searching for biomarkers of a wide range of socially significant diseases.

Approbation of work.

The main results of the study were presented at the "1st, 2nd and 3rd International Nanotechnology Forum" (Moscow, 2008-2010); "IV Congress of the Russian Society of Biochemists and molecular biologists”, Novosibirsk, 2008; at the International Congress "Human Proteome", Amsterdam, 2008; at the International Congress "Human Proteome", Sydney, 2010.

Publications.

The structure and scope of the dissertation.

The dissertation consists of an introduction, literature review, description of research materials and methods, research results and their discussion, conclusion, conclusions and list of references. The work is presented on 104 pages, illustrated with 33 figures and 4 tables, the list of references consists of 159 titles.

Analysis of scientific and technical groundwork in the field of highly sensitive proteomic technologies

One of the priority areas in modern science is to discover and elucidate the role of different types of proteins in the body, as well as understanding the molecular mechanisms that lead to the development of diseases.

Despite the constant improvement of proteomic methods, the number of newly discovered disease biomarkers remains almost / unchanged for last decade. This is due to the fact that the concentration limit of detection of traditional proteomic methods does not exceed 10"9 M. At the same time, it is important for proteomics to develop new analytical approaches for identifying proteins in a lower concentration range, in particular, low-copy protein molecules (with a concentration of 10"13 M and less), including biomarkers in biological material. Since it can be assumed that it is in these concentration ranges that the protein markers of most diseases are found.

One of the actively developing areas, which makes it possible to somewhat increase the concentration sensitivity of analysis, is the creation of analytical complexes based on nanochromatographic and nanoelectrophoretic systems compatible with mass spectrometers.

The nanochromatographic system in combination with mass spectrometry and electrospray type ionization made it possible to increase the sensitivity of protein detection by two orders of magnitude compared to chromatography high resolution(HPLC) . The concentration sensitivity limit of such conjugated systems is limited by the sensitivity of the electrophoresis/chromatography stage, and does not exceed 10-12 M for individual proteins (for example, for cytochrome C and bradykinin).

Currently, chromatographic methods have developed into separate independent areas - SELDI MS analysis (surface enhanced laser desorption and ionization/time of flight mass spectrometry), protein fishing methods using magnetic microparticles. In these technologies, the hydrophobic or charged surfaces of SELDI-chips are. or magnetic microparticles, combinations with mass spectrometric analysis, are successfully used: for? detection- and identification as separate types; proteins, and for protein/peptide profiling of blood serum [c, 8; \b, 15]. SEbDIi МЄ is: a powerful approach that allows you to study a biomaterial through the adsorption of biomolecules (proteins, peptides) on a chemically activated? surface (cation / anion exchange chips) followed by mass spectrometric analysis of adsorbed: molecules:. SEEDPMЄ approach is applied; for protein profiling of biomaterial; and recently: it has been used as a “diagnosis using proteomic barcodes” [17].. The essence of such a “barcode diagnosis” is to identify? features of the protein profile of the biological sample; associated with a particular disease: Yes, known; what d at. cancerous diseases, the “proteomic barcode” of the biomaterial is significantly different from that in healthy1 groups” of individuals: Therefore, control over changes in protein; composition of the biomaterial can become the basis for early diagnosis of diseases. On the; today, using the SELDI approach? МЄ markers were identified. gastric, ovarian, prostate, and breast cancers: A limitation of this method5 is the inability to identify proteins with high resolution and accuracy, which is especially important in; analysis of multicomponent mixtures such as biological material.

In addition to the problem of low concentration sensitivity of existing analytical systems, a stumbling block for the proteomic analysis of biological material has become a wide dynamic range of protein concentrations, especially in blood serum, which varies from 1(G M up to individual protein molecules. High-copy (major) proteins interfere in such systems detection and identification of low-copy (minor) proteins.

The problem of a wide concentration range of proteins in a biomaterial can be solved by applying methods for depleting blood serum from major protein fractions, methods for separating multicomponent mixtures and nanotechnological methods based on biospecific and chemical fishing of protein molecules of an analyte from complex mixtures on the surface of chips to various biosensors or on an activated surface. magnetic microspheres.

Traditionally, one-dimensional, more often two-dimensional gel electrophoresis is used to separate multicomponent protein mixtures. The principle of protein separation by two-dimensional gel electrophoresis is based on the difference between proteins according to the values ​​of their isoelectric points Hf of molecular weights. In proteomics, these approaches are used for protein mapping of biomaterial (tissue, blood plasma, etc.). The combination of 1D and/or 2D electrophoresis with mass spectrometry allows the identification of separated and visualized proteins. However, the procedure of two-dimensional gel electrophoresis is still not automated, it is rather complicated and time-consuming to perform, it requires a high qualification of the operator, and the analysis results are often poorly reproducible.

More convenient in comparison with two-dimensional electrophoresis, the procedure for separating proteins is high-performance chromatography (HPLC); which is an automated procedure that allows you to remove high-copy proteins from a complex mixture in order to subsequently identify low-copy proteins.

In order to directly identify proteins in complex mixtures, a chromatographic column can be connected to a mass spectrometer. However, intact proteins are practically not amenable to high-quality separation using HPLC, since they denature during analysis (due to low pH values ​​of the medium and high concentration of organic solvents), and also due to the low accuracy of mass spectrometric analysis, therefore, direct identification of most intact proteins , especially with a molecular weight exceeding 10 kDa, is often impossible. Analytical measurement accuracy can be improved by hydrolytic cleavage of proteins to peptide fragments, molecular weight from 700 to 4000 Da using proteases; such as trypsin (bottom-up technology). To achieve a qualitative separation of proteins in a mixture, a combination of several chromatographic procedures is used, the so-called multidimensional chromatography.

Methods for diagnosing hepatitis

Currently, for the protein diagnosis of hepatitis C, test systems for the detection of anti-HCVcore are used. The first ELISA tests detecting the presence of anti-HCVcore antibodies became available in the early 1990s, but they had low sensitivity and selectivity. Later, in the late 1990s, a new generation of anti-HCVcore ELISA tests appeared, which had a fairly high sensitivity of about 95-99% and could detect HCV several months after infection.

For example, in 1996, test systems developed by Vector-Best (Novosibirsk) and Diagnostic Systems (Nizhny Novgorod) appeared on the Russian market to detect antibodies - anti-HCV of the IgM class. The role of IgM antibodies in serodiagnosis has not been sufficiently studied, however, some studies have shown the importance of this marker for the detection of chronic hepatitis C. It has also been established that the correlation between the detection of viral RNA and anti-HCV IgM in patients is 80-95%. To determine the phase of development of viral hepatitis C Afanasyev A.Yu. et al. used a coefficient reflecting the ratio of anti-HCV IgG to anti-HCV IgM in the blood of patients. To date, many enzyme-linked immunosorbent assay (ELISA) systems have been developed that detect circulating antibodies to many epitopes of the hepatitis E virus.

Modern laboratory diagnostics of: viral hepatitis E in most medical institutions in Moscow, is carried out; in accordance with the existing orders of the Ministry of Health of the Russian Federation and the Department of Health: Moscow and is to determine immunoglobulins? class G to hepatitis E virus (anti-HGV IgG) in the blood serum of patients. Identification of this marker makes it possible to judge the presence of a current or past infection.

Disadvantages of the methods; EEISA-based detections, in addition to low sensitivity (more than GO "12 M)j, are also due to false-detection; viral hepatitis E in patients - due to post-infectious immunity,., cross-reactivity of antibodies, as well as insufficient sensitivity in the acute period) phase BFG BI LINKS: THIS is an active search for sensitive, specific, fast and easy-to-perform methods for detecting1 markers of “hepatitis E .

Another group of methods for detecting viral hepatitis in serum: blood is-B_registration; RNA BEI using PCR; Definition of RNA. BFG methods; GAD cannot be used as a primary test for - confirmation or exclusion; diagnosis; But; May be; Useful for confirming the diagnosis: Diagnosis of 1 BFG is by analysis of the 5' non-coding RNA region. However, the results of the analysis vary among different BFG genotypes.

Biological microchips have appeared on the Russian market, which make it possible to conduct - BFG genotyping and - determine an effective antiviral scheme; therapy. This biochip is an oligonucleotide chip for BFG genotyping based on the analysis of the NS5B region. The obtained results indicate the ability of the biochip to identify all 6 HCV genotypes and 36 subtypes, including the most virulent and drug-resistant forms.

On the one hand, PCR analysis methods are supersensitive and allow detecting and amplifying the signal from just one RNA molecule in a sample, but on the other hand, these methods are characterized by false positive results due to random contamination of samples, false negative results due to the high mutability of the virus and a relatively high analysis cost. Even in the same person, HCV RNA levels can periodically change by more than a millifold, leading to false negative results if low? virus replication or if the virus persists in tissues without entering the blood. The results of the quantitative determination of RIG, HCV in different laboratories do not agree well enough.

Of particular value for the early detection of viral hepatitis C Bt biomaterial are HCV protein antigens due to the fact that they appear1 in the blood serum several weeks earlier, even before the development of a full-fledged immune response of the body.

The HCVcoreAg surface antigen of the hepatitis C virus is the main marker of infection with the hepatitis C virus. It is detected 16 weeks before the appearance of antibodies in the blood due to the immune response of the body and before the development of clinical signs, while it is recorded both in the acute and chronic phases diseases. There is only one foreign commercial product (“Ortho Clinical Diagnostics”) for ELISA diagnostics of hepatitis C during the acute phase, based on the detection of HCVcoreAg.

The structural protein HCVcoreAg, consisting of 121 amino acid residues, is located at the N-terminus of the polypeptide and is formed under the influence of cellular proteases. The first proteolytic hydrolysis occurs between residues 191 and 192 (site C1) and leads to the formation of E1 glycoprotein. The second cleavage site (C2) is between amino acids 174 and 191. The corresponding cleavage products are named p21 and p23. Analysis of expression in a number of mammalian cells showed that p21 is the main product, while p23 is found in minor amounts. It is possible that cleavage at the C1 and C2 sites is an interrelated process, since p21 is formed under conditions when hydrolysis at G2 is not observed [D45]. HCVcoreAg is the main RNA-binding protein that appears to form the viral nucleocapsid. The biochemical properties of this protein are still poorly characterized. AFM studies of hepatitis C virus particles made it possible to obtain an image of the HCV capsid.

ASM chips

In the experimental part of the work, two types of AFM chips were used. The first type was used for MS identification of model proteins on the surface of AFM chips. These chips were substrates with functionally active chemical groups (hereinafter referred to as AFM chips with a chemically activated surface), on which the studied molecules were caught and irreversibly immobilized due to covalent bonds, the so-called “chemical fishing” procedure. The second type of AFM chips was used for MS identification on their surface of proteins biospecifically isolated from the analyte solution. Biological probes were previously immobilized on the surface of these chips - in the working areas. Monoclonal antibodies against marker proteins of viral hepatitis B and C (BFB and BFC) or aptamerr against the gpl20 protein and thrombin were used as biological probes. For biospecific-fishing procedures, chips with covalently immobilized probe molecules were incubated. in % analyte solution containing only detectable protein, or blood serum samples

To perform the task of MS identification of model proteins covalently immobilized on the surface of AFM chips of the first type, the following were used in the work: avidin (Agilent, USA), HSA (Agilent, USA), P450 VMZ (kindly provided by Professor A.V. Munro, University of Manchester, UK), thrombin (Sigma, USA), a-FP and anti-a-FP (USBio, USA); To perform the task of MS identification of proteins on the surface of AFM chips of the second type, biospecifically isolated from an analyte solution, monoclonal antibodies (MABs) were used as probe molecules: anti-HCVcore (Virogen, USA), anti-HBVcore (Research Institute of Molecular Diagnostics, Moscow), anti-HBsAg (Aldevron, USA), as target molecules: HBVcoreAg, HCVcoreAg (Virogen, USA) and HBsAg (Aldevron, USA), gpl20 (Sigma, USA), troponin (USBio, USA).

In addition, the following substances were used in the work: acetonitrile, isopropanol, formic acid, distilled water (Merck, USA), trifluoroacetic acid (TFA), ammonium bicarbonate (Sigma, USA), a-cyano-4-hydroxycinnamic acid (HCCA), dihydroxybenzoic acid-(DHB) (Bruker Daltonics, Germany), trypsin (Promega, USA).

Blood serum samples for AFM study were provided by the Department of Infectious Diseases in Children of the Russian State Medical University, Central Research Institute of Epidemiology of Rospotrebnadzor, MNIIEM "n. Gabrichevsky: The presence of hepatitis C virus (HCV) particles in blood serum samples was confirmed using the polymerase chain reaction (PCR) method using the test system "Amplisens HCV Monitor" (Central Research Institute of Epidemiology of the Ministry of Health of the Russian Federation, Moscow).

AFM analysis was carried out at the Laboratory of Nanobiotechnology, IBMC RAMS. The calculation of proteins and antigen/antibody complexes on the surface of the AFM chip was carried out on the basis of the correlation of the heights of the corresponding images of proteins and their complexes, measured using AFM, according to the method described in . Was used by ACM NTEGRA (NT-MDT, Russia). AFM measurements were carried out in the semi-contact mode. NSG10 series cantilevers from NT-MDT were used as probes. The typical radius of curvature of the needles was 10 nm, and the resonant frequency ranged from 190 to 325 kHz. The chip scanning area was 400 µm2. Each measurement was carried out at least 3 times.

Immobilization of proteins and aptamers on the surface of the AFM chip was carried out according to the following procedure.

To a protein solution (0.1 µM) with a volume of 2 µl was added 8 µl of a solution of a mixture of NHS/EDC (v/v=l/l) and thoroughly mixed. The resulting mixture was applied to the surface of the silanized chip and incubated for 2 minutes at room temperature. The chip was then washed twice in a thermoshaker with 1 ml of deionized water at 800 rpm and 37°C. The quality of protein immobilization on the surface of the AFM chip was monitored by atomic force microscopy.

Immobilization of aptamers on the chemically activated surface of the AEM chip was carried out as follows. To a stock solution of DSP at a concentration of 1.2 mM in DMSO/ethanol (v/v=l/l)4 was added a solution of PBS buffer 50 mM (pH 7.4) also in a ratio of 1/1 by volume. The working solution thus obtained was applied to the surface of the AFM chip and incubated for 10 minutes. After that, washing was carried out with a 50% solution of ethanol in water with a volume of 1 ml at 15C for 10 minutes. An aptamer solution with a concentration of 3 JIM was applied to the activated zone of the AFM chip and incubated for 4 minutes with stirring at a speed of 800 rpm. Blocking of unreacted amino groups of the DSP cross-linker was carried out in the presence of 5 mM Tris-HCl solution for 10 minutes at 37°C. The final washing step was carried out twice aqueous solution volume of 1 ml for 10 minutes at 25C.

A trypsinolytic mixture containing a buffer solution of 150 mM NH4HCO3, acetonitrile, 0.5 M guanidine hydrochloride, and glycerol (pH 7.4) was applied to the surface of the AFM chip with immobilized probe molecules. Then, 0.5 μl of modified porcine trypsin solution at a concentration of 0.1 μM was added to the buffer solution. The AFM chip was incubated in a humid environment for 2 hours at a constant temperature of 45C, 0.5 µl of trypsin solution (0.1 µM) was again added to its surface, and the incubation continued for another 12 hours. The trypsinolytic mixture was washed off the surface of the AFM chip with a 10 µl elution solution containing 70% acetonitrile in 0.7% trifluoroacetic acid (TFA). The hydrolyzate thus obtained from the surface of the AFM chip was dried in a vacuum evaporator at 45°C and 4200 rpm. Next, the peptide mixture was dissolved in 10 µl of a 5% formic acid solution or in 10 µl of a 0.7% TFA solution for subsequent MS analysis.

During MS analysis with the MALDI type of ionization, the samples were prepared as follows. Samples dissolved in 0.7% TFA solution with a volume of 10 µl were concentrated and desalted using ZipTip C18 microtips (Millipore, USA) according to the manufacturer's protocol and mixed with a saturated solution of a matrix containing HCCA or DHB in 50% acetonitrile solution with 0 .7% TFA. The resulting mixture was applied to an MTP size MALDI target.

-identification of proteins caught by "chemical fishing" on the surface of an AFM chip from an analyte solution

On this stage experimental work MS spectra were obtained for model proteins chemically immobilized on the surface of AFM chips from an analyte solution. The range of concentrations of the studied proteins in the analyte solution for avidin, HSA, anti-aFP was 10"-10"9 M, troponin, aFP and P450 VMZ - 10"6-10"8 M.

MS analysis was carried out for 6 types of proteins, different in their origin, molecular weight, the number of trypsinolysis sites and their spatial accessibility, the degree of hydrophobicity of the amino acid sequence (the ratio of hydrophobic amino acids to hydrophilic ones), which were covalently immobilized on the surface of the AFM chip from the analyte solution ( Table 1). In these experiments, AFM chips were used, which contained the working and control zones. The working zone was a chemically activated area of ​​the AFM chip surface, on which the model proteins were “chemically fished”; the control zone was the chemically inactive region of the chip surface. The count of visualized captured molecules was recorded using AFM. The experimental data of AFM analysis obtained for the above model proteins, namely the number of molecules caught on the surface of the working area of ​​the AFM chip, are presented in Table 2. protein in analyte solution.

As can be seen from Table 2, the number of molecules registered in the working area of ​​the AFM chip for all presented proteins was -1040 molecules. The sensitivity limit of MS detectors is about 105 molecules. Thus, for the presented model proteins, successful irreversible immobilization on the surface of the AFM chip was carried out, and the number of AFM-registered protein objects was sufficient for subsequent MS identification. At the same time, the minimum recorded concentration of model proteins in the incubation solution was quite low, 10"-10" M.

Mass spectrometric analysis of the samples was performed using MALDI and ESI types of ionization. AFM chip after incubation in the appropriate solution of avidin with a concentration of 10"9 M. Analysis of these spectra made it possible to reliably identify avidin (Gallus Gallus) by its two proteotypic peptides: SSVNDIGDDWK (m/z=618.6) and VGINIFTR (m/z= 460.4). Both peptides had well-defined peaks of their doubly charged ions (MS-spectra). Using AFM-MS analysis of the chemically activated working zone of the AFM-chip after incubation in a solution of the analyte protein with a concentration of 10"8 M, another small protein was detected - troponin I. The MS and MS/MS spectra corresponding to the peptide doubly charged ion 1449 Da are shown in Figure 3. MS analysis of the experimentally obtained spectra made it possible to reliably identify and identify human troponin (gi 2460249) on the surface of the AFM chip with a probability of more than 95% .

Figure 5 shows tandem fragmentation spectra of a globular protein, human serum albumin (HSA), which performs transport functions in blood plasma. The spectra were obtained from the chemically activated working zone of the AFM chip after incubation in an appropriate albumin solution with a concentration of 10"9 M. The analysis of these spectra made it possible to reliably identify human albumin by its two proteotypic peptides: VPQVSTPTLVEVSR (m/z=756.5) and YLYEIAR (m/z=464.3) Both peptides had well defined peaks of their doubly charged ions (MS spectra).

MS/MS spectra of trypsinized objects from the chemically activated surface of the AFM chip incubated in a solution of human serum albumin (C=10 9 M). VPQVSTPTLVEVSR peptide with m/z=756.5 (A), YLYEIAR peptide with m/z=464.3 (B). Experimental conditions: measurements were carried out on an LC/MSD Trap XCT Ultra mass spectrometer (Agilent).

Thus, MS analysis made it possible to identify proteins detected using AFM. Based on the data obtained, a relationship was revealed between the number of identified proteotypic peptides on the surface of the AFM chip and the content of the desired protein in the analyte solution. Such a dependence, for example, for the P450 VMZ and HSA proteins covalently immobilized on the chemically activated surface of the AFM chip, is shown in Figure 6. As can be seen in Figure 6, the higher the protein concentration in the analyte solution (-KG6 M), the greater the number of peptides can be reliably identified both in the case of MALDI-MS and ESI-MS analysis. Significant differences between the number of identified peptides in the concentration range of 10"6-10"9 M among the analyzed proteins in the analyte solution were not observed.

Dependences of the number of identified peptides of analyte molecules on the protein concentration in the incubation solution. (A) - analysis of a mixture of peptides of model proteins HSA, VMZ on mass spectrometers with MALDI-type ionization Bruker Microflex (Bruker Daltonics, Germany) and Autoflex III (Bruker Daltonics, Germany); (B) - analysis of a mixture of peptides of model proteins HSA, VMZ on a mass spectrometer with ESI-type ionization LC/MSD Trap XCT Ultra (Agilent, USA).

The results obtained made it possible to conclude that AFM-MS (MALDI and ESI) makes it possible to detect and identify protein molecules covalently extracted from the analyte solution on the surface of the AFM chip, which differ in their physicochemical properties.

At the same time, in the control zone of the AFM chip (non-activated) after its incubation in the analyte solution, the AFM method did not detect the presence on the chip surface of objects corresponding in height to protein molecules. MS analysis also did not reveal objects of a protein nature. Thus, it was experimentally proved that AFM adequately registers the desired objects - protein molecules of the analyte.

The next stage of this work was the development of an AFM-MS combination scheme for the identification of proteins recovered from a solution of za. through biospecific interactions.

The scheme of mass spectrometric analysis in the case of biospecific AFM fishing of proteins from a solution is shown in Figure 7. According to the above scheme, probe molecules were first immobilized on the surface of the working area of ​​AFM chips, which were monoclonal1 antibodies against protein markers of viral hepatitis B and C or aptamers against proteins of the HIV-1 glycoprotein gpl20 and thrombin, while the surface of the control zone did not contain immobilized probe molecules. The quality control of the immobilization of probe molecules was carried out by AGM-visualization. Then, such a chip was incubated in an analyte solution containing the protein under study. After the stage of washing off nonspecifically adsorbed molecules on the chip surface, and the stage of sample preparation for subsequent mass spectrometric analysis on the AFM chip surface, MS analysis of the AFM-detected proteins was performed.

The experimental part of this section involved two stages of analysis. At the first stage, it was necessary to carry out the MS identification of protein probe molecules covalently immobilized on the AFM chip, and at the second stage, target proteins caught on the corresponding partner molecules from a solution or from blood serum samples due to biospecific interactions. For this purpose, MS analysis of MCA covalently immobilized on the surface of AFM chips against HCV and HBV marker proteins: anti-HCVcore and anti-HBVcore was performed. For mAbs against anti-HCVcore and anti-HBVcore proteins, tandem fragmentation spectra and peptide map spectra were obtained in this work for the first time.

high storage capacity, which guarantees their active biological principle.

LITERATURE

1. Klychkova G.Yu. Development of technology for a complex preparation from the cartilage tissue of squid, salmon and sturgeon // Materials of Vseros. Internet conf. young scientists. - Vladivostok: TIN-RO-Center, 2004. - S. 164-170.

2. Metzler D. Biochemistry. T. 2. - M., 1980. - 605 p.

3. Sukhoverkhova G.Yu. Biochemical characteristics of the cartilaginous tissue of hydrobionts and the technology of dietary supplements for food: Dis. ... cand. tech. Sciences. - Vladivostok, 2006. - 157 p.

4. Sytova M.V. Scientific substantiation of the complex processing of Amur sturgeon fish: Abstract of the thesis. dis. ... cand. tech. Sciences

M.: VNIRO, 2005. - 24 p.

Department of Food Biotechnology

Received 07.02.07

IDENTIFICATION OF PROTEIN COMPONENTS OF KERATIN ENZYMATIC HYDROLYZATE

Ch.Yu. SHAMKHANOV, L.V. ANTIPOVA

Grozny State Oil Institute Voronezh State Technological Academy

One of the most effective ways of processing secondary resources of the meat and poultry processing industry is the use of modern biotechnology methods to obtain food hydrolysates. The functional and technological properties of the obtained products depend on the biochemical composition and molecular weight of its protein components.

When using physicochemical methods to determine the molecular weight of proteins, the result depends not only on the mass, but also on the electric charge and shape of the protein molecule, especially when changing the rate of protein diffusion, the rate of sedimentation in a gravitational field. In this regard, when determining the molecular weights of proteins, it is preferable to use statistical methods when the protein solution is in equilibrium, for example, when passing it through a column filled with gel.

The purpose of the work is to determine the molecular weight M of the protein components of the keratin enzymatic hydrolyzate and its biochemical composition.

The gel filtration method was used to determine the molecular weight of the protein components of the keratin hydrolyzate. Sephadex v-100 (medium, particle diameter 40-120 µm) was used with fractionation limits of 4000-150000 Da.

A 46.0 x 1.9 cm column was filled with Sephadex treated with 0.02 M universal buffer, pH 7.0. 1.5 cm3 of a solution of -7 mg/cm3 - keratin hydrolyzate was applied to it and eluted with the same universal buffer at a rate of 12 cm3/h. Fractions of 3 cm3 were collected and then the protein content in them was determined spectrophotometrically on SF-46 at 280 nm. To determine the molecular weight of the keratin hydrolyzate fractions, the column with Sephadex was pre-calibrated under the same conditions using several pure (marker) proteins with known M. The calibration curve was built using a linear relationship between ^ M and volume

eluate Ye, released from the column. In table. 1 shows some of the physicochemical characteristics of marker proteins.

Table 1

Marker protein M, Yes 1§ m V, cm3

Lysozyme 13930 4.143 54

Trypsin 25700 4.409 48

Peroxidase 34000 4.531 45

Bovine albumin 68000 4.832 36

Blue dextran 2000000 6.301 21

Water soluble fraction

keropeptide<10000 2,845 72

The water-soluble fraction of the keratin hydrolyzate leaves the column in a much smaller volume than the marker protein lysozyme with the lowest molecular weight. Therefore, in the keratin hydrolyzate (keropeptide) there are no protein fractions with M > 13930 Da. The approximate mass of hydrolysis products is below 10,000 Da, determined by gel filtration on Sephadex B-100 marker proteins. Linear dependency between ^ M of proteins and the volume of the eluate Ye, released from the column, is shown in fig. 1 (1 - blue dextran; 2 - bovine albumin; 3 - peroxidase; 4 - trypsin; 5 - lysozyme; 6 - water-soluble fraction of keropeptide).

Due to the absence of marker proteins in the studied range of 10000-5000 Da, the search for the water-soluble protein fraction was carried out using porous

table 2

M, Yes Soluble protein and peptides, mg/cm3 Total peptides and amino acids, µg/cm3 Tyrosine, µmol/cm3 Reducing substances, µg/cm3

0-10000 5,64 7337 7,835 2815

0-5000 4,21 5278 5,960 2272

% of baseline 74.6 71.9 76.1 80.7

membranes of grades UPM-100 and UAM-50 on a laboratory ultrafiltration unit (CJSC NPO Tekhkon). The scheme included the installation itself with a 5% solution of keropeptide placed on a magnetic stirrer for its constant mixing. For efficient separation of the protein solution, compressed air was supplied to the upper part of the apparatus under pressure. The hydrolysis products passed through porous membranes, alternately replaced depending on the desired molecular weight of the ultrafiltrate, to the lower part of the apparatus and collected in a receiving vessel. In the resulting ultrafiltrates, a number of biochemical parameters were determined, which made it possible to evaluate the distribution of hydrolysis products by molecular weight (Table 2).

The keropeptide contains low molecular weight proteins with M 5000-10000 Da. Their mass fraction is estimated as the difference in readings for fractions 0-10000 and 0-5000 Da and is 1.43 mg/cm3. This fraction also gave a positive reaction to the ninhydrin reaction (2059 µg/cm3), tyrosine (1.875 µmol/cm3) and reducing substances (543 µg/cm3). However, the main share of the hydrolysis products is concentrated in the lower molecular weight fraction with М< 5000 Да, соответствующей по существующей классификации фракции пептидов. Массовая доля всех исследуемых показателей составляет более 7 0% от аналогичных значений во фракции 0-10000 Да.

Further determination of the molecular weight of water-soluble protein fractions of the keropeptide was carried out on Sephadex B-25 (average, particle diameter 50-150 μm), which allows it to be set within the fractionation range of 1000-5000 Da. The gel filtration conditions remained unchanged. By

The results of protein determination in the fractions were used to construct an elution profile. In all fractions, in addition to protein, the content of low molecular weight substances was determined by the ninhydrin method, tyrosine, and reducing substances (RS), and the quantitative distribution of protein fractions was also determined. On fig. Figure 2 shows a gel chromatogram of an enzymatic keratin hydrolyzate through Sephadex B-25 (curve 1 - protein; 2 - ninhydrin test; 3 - tyrosine; 4 - PB).

In this case, the protein is detected in the form of two small peaks almost in the very first fractions. Mass fraction of protein in fractions No. 1-8 from 3-24 cm3

has rather high values ​​and amounts to 0.12-0.18 mg/cm3 (curve 1).

The bulk of the product came out of the column as the maximum protein peak with a volume of 27 cm3 of eluate (fraction No. 9, 3 cm3 of eluate each). The mass fraction of protein in this fraction was registered at the level of 0.66 mg/cm3, which is 3-4 times higher than in the other studied fractions.

Further elution of the keropeptide in the volume range of 36-96 cm3 revealed three peaks with a decrease in the mass fraction of protein in them by no more than 0.08 mg/cm3. The complete keropeptide solution is eluted in a final volume of 96 cm3.

In fraction no. 9, the entire amount of available RS, mass fractions of 66 μg/cm3, was found (curve 4). The ninhydrin reaction was used in gel chromatography to identify the distribution of hydrolysis products by molecular weight. It has been established that when an excess amount of ninhydrin and protein products interact with a free MH2 group, and depending on the number of these groups, it is possible to locate the protein

RV, μg/cm3 175 co D 5 o l s; .7 0

100 125 X - 3 co o o. 0.5

80 § 100 2 _ n 0.4

60 1 isin, 7 sl 0.3

40 1 l 5 o - (P 2 o I- 0.2

20 25 _ 2 ai O 0.1

6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 V, cm

derivatives by elution through Sephadex. Thus, a low mass fraction of ninhydrin (4–6 µg/cm3) very accurately reflects the amount of free amino groups and determines the presence of high molecular weight peptides with М 3000–5000 Da in fractions J# 1–8 (curve 2). It is known that in the range of measuring the molecular weight of hydrolysis products< 5000 Да входят только пептиды и аминокислоты. При больших концентрациях белка (фракция № 9) соответственно увеличивается окрашивание его свободных аминогрупп нингидрином -64 мкг/см3. Этот диапазон элюции характеризует наличие во фракциях № 8-12 среднемолекулярных пептидов с М 300-3000 Да. Определяемый с помощью маркерных белков по калибровочной кривой lg M керопептида 2,845 также указывает на его ориентировочную массу >700 Yes. Further analysis of the elution profile for the ninhydrin sample (fractions nos. 12-36) revealed a peak that did not correspond to its protein concentration, as was observed for peptides in previous fractions. The mass fraction of low molecular weight substances in fraction No. 23 was 157 µg/cm3, which is more than 2 times higher than that for peptides in fraction No. 9. the presence of hydrolysis products in the form of free amino acids in the last fraction. Belonging to it and the amino acid tyrosine (0.06 µmol/cm3) is another proof of the stated position.

Thus, gel filtration of keratin hydrolyzate through Sephadex G-100 and G-25 indicates the presence of a low molecular weight solute in it.

my protein (< 10000 Да), составляющего 25% от его общего количества в гидролизате. Преобладающая часть белковых продуктов - 75% - представлена среднемолекулярными пептидами (300-3000 Да) с ориентировочной М >700 Yes. In this case, protein chains contain 5-20 amino acid residues. It is important to emphasize that fraction No. 9 simultaneously gives reactions to the biuret bond, ninhydrin, tyrosine, and RV. This characteristic suggests the presence of carbohydrates in the keratin protein and their direct connection with the protein as part of a single complex.

LITERATURE

1. Antipova L.V., Pashchenko L.P., Shamkhanov Ch.Yu., Kurilova E.S. Obtaining and characterization of food keratin hydrolyzate // Storage and processing of agricultural raw materials. - 2003. - No. 7.

2. Antipova L.V., Shamkhanov Ch.Yu., Osminin O.S., Pozhalova N.A. Biochemical characteristics of the process of enzymatic hydrolysis of keratin-containing raw materials in the poultry processing industry. Izv. universities. Food technology. - 2003. - No. 5-6.

3. Antipova L.V., Shamkhanov Ch.Yu., Osminin O.S. Co -

improvement of technology for the production of keropeptide from feather-down raw materials // Meat industry. - 2004. - No. 3. - S. 44-47.

4. Rogov I.A., Antipova L.V., Dunchenko N.I., Zhereb-tsov N.A. Chemistry of food. In 2 books. Book. 1. Proteins: structure, functions, role in nutrition. - M.: Kolos, 2000. - 384 p.

5. Osterman L.A. Chromatography of proteins and nucleic acids. - M.: Nauka, 1985. - 536 p.

6. Kochetov G.A. A practical guide to enzymology. - 2nd ed., revised. and additional - M.: Higher. school, 1980. - 272 p.

Department of Food Technology Department of Technology of Meat and Meat Products

Received 0S.02.0? G.

THEORETICAL SUBSTANTIATION OF THE MECHANISM OF THE PRESERVING ACTION OF THE COMPONENTS OF SMOKING EXTRACTS

S.V. ZOLOTOKOPOVA, I.A. PALAGINA

Astrakhan State Technical University Astrakhan Branch of Saratov State Socio-Economic University

An important area of ​​research in recent years is the study of the effect of various food additives not only on taste and aroma, but also on increasing the shelf life of food products. Since ancient times, spicy plants have been used for canning, table salt, smoking, etc. The analysis shows that currently the market is conquered by smoke extracts. Smoked-flavored foodstuffs are especially popular among the population, and traditional smoking is losing its positions to smokeless.

Environmentally beneficial and promising are extracts obtained under gentle conditions.

G.I. has been working on improving the extraction technology for decades. Kasyanov - Honored Worker of Science and Technology of the Russian Federation, Honored Inventor of the Russian Federation, Doctor of Technical Sciences, Professor, Head of the Department of Technology of Meat and Fish Products of the Kuban State technological university. The scientific and pedagogical school "Theory and practice of processing raw materials of plant and animal origin with liquefied and compressed gases" operating at KubGTU and the Krasnodar Research Institute for the Storage and Processing of Agricultural Products under his leadership deals with the problems of increasing the efficiency of processing various raw materials, which make it possible to improve the quality of products, reduce processing times and at the same time reduce energy costs.

salting out: precipitation with salts of alkali, alkaline earth metals (sodium chloride, magnesium sulfate), ammonium sulfate; at the same time, the primary structure of the protein is not disturbed;

precipitation: use of dewatering agents: alcohol or acetone at low temperatures (about -20°C).

When using these methods, proteins lose their hydration shell and precipitate in solution.

Denaturation- violation of the spatial structure of proteins (the primary structure of the molecule is preserved). It can be reversible (the protein structure is restored after the removal of the denaturing agent) or irreversible (the spatial structure of the molecule is not restored, for example, when proteins are precipitated with concentrated mineral acids, salts of heavy metals).

Protein separation methods Separation of proteins from low molecular weight impurities

Dialysis

A special polymer membrane is used, which has pores of a certain size. Small molecules (low molecular weight impurities) pass through the pores in the membrane, while large molecules (proteins) are retained. Thus, proteins are washed from impurities.

Separation of proteins by molecular weight

Gel chromatography

The chromatographic column is filled with gel granules (Sephadex), which has pores of a certain size. A mixture of proteins is added to the column. Proteins, the size of which is smaller than the size of the Sephadex pores, are retained in the column, as they “get stuck” in the pores, and the rest freely leave the column (Fig. 2.1). The size of a protein depends on its molecular weight.

Rice. 2.1. Separation of proteins by gel filtration

Ultracentrifugation

This method is based on different rates of sedimentation (precipitation) of protein molecules in solutions with different density gradients (sucrose buffer or cesium chloride) (Fig. 2.2).

Rice. 2.2. Separation of proteins by ultracentrifugation

electrophoresis

This method is based on different rates of migration of proteins and peptides in an electric field depending on the charge.

Gels, cellulose acetate, agar can serve as carriers for electrophoresis. The molecules to be separated move in the gel depending on their size: those that are larger will be held back as they pass through the pores of the gel. Smaller molecules will encounter less resistance and therefore move faster. As a result, after electrophoresis, larger molecules will be closer to the start than smaller ones (Fig. 2.3).

Rice. 2.3. Separation of proteins by gel electrophoresis

Proteins can also be separated by electrophoresis by molecular weight. For this use electrophoresis in PAAG in the presence of sodium dodecyl sulfate (SDS-Na).

Isolation of individual proteins

Affinity chromatography

The method is based on the ability of proteins to bind strongly to various molecules by non-covalent bonds. Used to isolate and purify enzymes, immunoglobulins, receptor proteins.

Molecules of substances (ligands), with which certain proteins specifically bind, are covalently combined with particles of an inert substance. A mixture of proteins is added to the column, and the desired protein is firmly attached to the ligand. The remaining proteins freely exit the column. The retained protein can then be washed from the column with a buffer containing the free ligand. This highly sensitive method allows very small amounts of pure protein to be isolated from a cell extract containing hundreds of other proteins.

Isoelectric focusing

The method is based on different IEP values ​​of proteins. Proteins are separated by electrophoresis on a plate with ampholine (this is a substance that has a pre-formed pH gradient in the range from 3 to 10). During electrophoresis, proteins are separated according to the value of their IEP (in IEP, the charge of the protein will be zero, and it will not move in the electric field).

2D electrophoresis

It is a combination of isoelectric focusing and electrophoresis with SDS-Na. First, electrophoresis is carried out in a horizontal direction on a plate with ampholine. Proteins are separated depending on the charge (CEP). Then the plate is treated with a solution of SDS-Na and electrophoresis is carried out in the vertical direction. Proteins are classified based on molecular weight.

Immunoelectrophoresis (Western blot)

An analytical method used to determine specific proteins in a sample (Figure 2.4).

Isolation of proteins from biological material.

Separation of proteins by molecular weight by electrophoresis in PAAG with SDS-Na.

Transfer of proteins from the gel to the polymer plate in order to facilitate further work.

Treatment of the plate with a non-specific protein solution to fill the remaining pores.

Thus, after this stage, a plate was obtained, the pores of which contain separated proteins, and the space between them is filled with a nonspecific protein. Now we need to identify whether among the proteins we are looking for, responsible for some kind of disease. Antibody treatment is used for detection. Under primary antibodies understand antibodies to the desired protein. By secondary antibodies is meant antibodies to primary antibodies. An additional special label (the so-called molecular probe) is added to the composition of secondary antibodies, so that later the results can be visualized. Radioactive phosphate or an enzyme tightly bound to the secondary antibody is used as a label. Binding first to primary and then to secondary antibodies has two goals: to standardize the method and to improve results.

Processing with a solution of primary antibodies  binding occurs in the place of the plate where there is an antigen (the desired protein).

Removal of unbound antibodies (washing).

Treatment with a solution of labeled secondary antibodies for subsequent development.

Removal of unbound secondary antibodies (washing).

Rice. 2.4. Immunoelectrophoresis (Western blot)

In the case of the presence of the desired protein in the biological material, a band appears on the plate, indicating the binding of this protein to the corresponding antibodies.

The most characteristic physicochemical properties of proteins are: high viscosity of solutions, slight diffusion, the ability to swell over a wide range, optical activity, mobility in an electric field, low osmotic pressure and high oncotic pressure, the ability to absorb UV rays at 280 nm (this the latter property, due to the presence of aromatic amino acids in proteins, is used to quantify proteins).

Proteins, like amino acids, are amphoteric due to the presence of free NH2 and COOH groups and are characterized, respectively, by all the properties of acids and bases.

Proteins have pronounced hydrophilic properties. Their solutions have a very low osmotic pressure, high viscosity and little diffusivity. Proteins are capable of swelling to a very large extent.

The rad is associated with the colloidal state of proteins. characteristic properties, in particular the phenomenon of light scattering, which underlies the quantitative determination of proteins by nephelometry. This effect is also used in modern methods, microscopy of biological objects. Protein molecules are not able to pass through semi-permeable artificial membranes (cellophane, parchment, collodion), as well as biomembranes of plant and animal tissues, although with organic lesions, such as kidneys, the capsule of the renal glomerulus (Shumlyansky-Bowman) becomes permeable to blood serum albumins, and they appear in the urine.

Protein denaturation Under the influence of various physical and chemical factors, proteins undergo coagulation and precipitate, losing their native properties. Thus, denaturation should be understood as a violation of the general plan - the unique structure of a native protein molecule, leading to the loss of its characteristic properties (solubility, electrophoretic mobility, biological activity, etc.). Most proteins denature when heated with a solution above 50-60 ° C. External manifestations of denaturation are reduced to a loss of solubility, especially at the isoelectric point, an increase in the viscosity of protein solutions, an increase in the amount of free functional SH-rpypp and a change in the nature of X-ray scattering. The most characteristic sign of denaturation is a sharp decrease or complete loss by the protein of its biological activity (catalytic antigenic or hormonal). peptide bonds of the very backbone of the polypeptide chain At the same time, globules of native protein molecules unfold and random and disordered structures are formed.