The required daily protein intake leads to muscle tissue nutrition and proper amino acid levels. Symptoms of an excess of protein in the body indicate tissue poisoning with the products of its decay, which gives the patient internal and external discomfort.

Protein in the body - what is it?

Amino acids, interconnected in a special way, form high-molecular organic compounds in the body - proteins. In an unchanged form, the protein that enters the body is not absorbed, therefore it is broken down into amino acids.

In the body, the necessary proteins are formed from amino acids, which perform a number of functions:

• Compounds are a constituent part of the organelles and cytoplasm of the cells of the body. For example, connective tissue protein is involved in the growth of hair, nail plates, tendons, etc.

Protein in the body plays an important role in the normal functioning of all organs. Therefore, it is very important to control your protein intake. An overabundance of protein very often leads to serious illnesses, therefore, at the first signs of a deviation, it is necessary to consult with specialists.

Protein(synonym proteins) - high-molecular nitrogenous organic compounds that are polymers of amino acids. Proteins are the main and essential component of all organisms.

The dry matter of most organs and tissues of humans and animals, as well as most microorganisms, consist mainly of proteins. Protein substances underlie the most important vital processes. So, for example, metabolic processes (digestion, respiration, excretion, etc.) are provided by the activity of enzymes (see), which are proteins by their nature. Proteins also include the contractile structures underlying the movement, for example, the contractile protein of muscles (actomyosin), the supporting tissues of the body (collagen of bones, cartilage, tendons), the integuments of the body (skin, hair, nails, etc.), which are the main way from collagens, elastins, keratins, as well as toxins, antigens and antibodies, many hormones and other biologically important substances.

The role of proteins in a living organism is emphasized by their very name "proteins" (Greek protos first, primary) proposed by Mulder (G. J. Mulder, 1838), who discovered that the tissues of animals and plants contain substances that resemble egg white in their properties. It was gradually established that proteins are a vast class of diverse substances, built according to the same plan. Noting the paramount importance of proteins for vital processes, Engels determined that life is a way of existence of protein bodies, which consists in the constant self-renewal of the chemical constituent parts of these bodies.

Chemical composition and structure of proteins

Proteins contain on average about 16% nitrogen. With complete hydrolysis, proteins decompose with the addition of water to amino acids (see). Protein molecules are polymers that consist of residues of about 20 different amino acids belonging to the natural L-series, that is, having the same configuration of the alpha carbon atom, although their optical rotation may be unequal and not always directed in the same direction. The amino acid composition of different proteins is not the same and serves as the most important characteristic of each protein, as well as a criterion for its value in nutrition (see the section Proteins in nutrition). Some proteins may be devoid of certain amino acids. For example, maize proteins - zein does not contain lysine and tryptophan. Other proteins, on the other hand, are very rich in individual amino acids. Thus, salmon protamine - salmin contains over 80% arginine, silk fibroin - about 40% glycine (the amino acid composition of some proteins is presented in Table 1).

Table 1. AMINO ACID COMPOSITION OF SOME PROTEINS (in weight percent of protein amino acids)

With incomplete (usually enzymatic) hydrolysis of proteins, in addition to free amino acids, a number of substances with relatively low molecular weights are formed, called peptides (see) and polypeptides. In proteins and peptides, amino acid residues are interconnected by the so-called peptide (acid-amide) bond formed by the carboxyl group of one amino acid and the amino group of another amino acid:

Depending on the number of amino acids, such compounds are called di-, tri-, tetrapeptides, etc., for example:

Long peptide chains (polypeptides), consisting of tens and hundreds of amino acid residues, form the basis of the structure of a protein molecule. Many proteins consist of a single polypeptide chain, while other proteins have two or more polypeptide chains interconnected to form a more complex structure. Long polypeptide chains of the same amino acid composition can produce a huge number of isomers due to the different sequence of individual amino acid residues (just as many different words and their combinations can be made from 20 letters of the alphabet). Since different amino acids can be included in the composition of polypeptides in different ratios, the number of possible isomers becomes almost infinite, and for each individual protein the amino acid sequence in the polypeptide chains is characteristic and unique. This amino acid sequence determines the primary structure of the protein, which in turn is determined by the corresponding sequence of deoxyribonucleotides in the structural genes of the DNA of a given organism. To date, the primary structure of many proteins has been studied, mainly protein hormones, enzymes, and some other biologically active proteins. The sequence of amino acids is determined by enzymatic hydrolysis of beks and obtaining the so-called peptide maps using two-dimensional chromatography (see) and electrophoresis (see). Each peptide is analyzed for terminal amino acids before and after treatment with aminopolypeptidase, a specific enzyme that sequentially cleaves amino-terminal (N-terminal) amino acids, and carboxypolypeptidase, which cleaves carboxy-terminal (C-terminal) amino acids. To determine the N-terminal amino acids, reagents are used that combine with the free amino group of the terminal amino acid. Usually dinitrofluorobenzene (1-fluoro-2,4-dinitrobenzene) is used, which gives a dinitrophenyl derivative with an N-terminal amino acid, which can then be identified after hydrolysis and chromatographic separation of the hydrolyzate. Along with dinitrofluorobenzene, proposed by F. Sanger, treatment with phenylisothiocyanate according to P. Edman is also used. In this case, phenylthiohydantoin is formed with the terminal amino acid, which is easily cleaved from the polypeptide chain and can be identified. To determine the C-terminal amino acids, heating of the peptide in acetic anhydride with ammonium thiocyanate is used. As a result of condensation, a thiohydantoin ring is obtained, which includes a terminal amino acid radical, which can then be easily cleaved from the peptide and establish the character of the C-terminal amino acid. The sequence of amino acids in a protein is determined based on the sequence of peptides obtained using different enzymes and taking into account the specificity of each enzyme that cleaves a protein at a peptide bond formed by a particular amino acid. Thus, the determination of the primary structure of a protein is a very painstaking and time-consuming work. Various methods of direct determination of the amino acid sequence by means of X-ray structural analysis (see) or by mass spectrometry (see) of peptide derivatives obtained by hydrolysis of protein by different enzymes have been successfully applied.

Spatially polypeptide chains often form helical configurations held by hydrogen bonds and forming a secondary structure of the protein. The most common is the so-called a-helix, in which there are 3.7 amino acid residues per turn.

Separate amino acid residues in the same or in different polypeptide chains can be connected to each other using disulfide or ether bonds. Thus, in the insulin monomer molecule (Fig. 1), the 6th and 11th cysteine ​​residues of the A-chain and the 7th and 20th cysteine ​​residues of the A-chain, respectively, with the 7th and 19th cysteine ​​residues of the B-chain are interconnected by disulfide bonds. Such bonds give the polypeptide chain, which usually has helical and non-helical regions, a certain conformation, called the tertiary structure of the protein.

Rice. 1. Diagram of the amino acid sequence in the bovine insulin monomer molecule. Above - chain A, below - chain B. Bold lines indicate disulfide bonds; in circles - abbreviated names of amino acids.

The quaternary structure of a protein is understood to mean the formation of complexes from monomeric protein molecules. For example, a hemoglobin molecule consists of four monomers (two alpha chains and two beta chains). The quaternary structure of the enzyme lactate dehydrogenase is a tetramer consisting of 4 monomeric molecules. These monomers are of two types: H, which is characteristic of the heart muscle, and M, which is characteristic of skeletal muscle. Accordingly, there are 5 different isoenzymes of lactate dehydrogenase, which are tetramers from different combinations of these two monomers - HHHH, HHHM, HHMM, HMMM and MMMM. The structure of a protein determines its biological properties, and even a slight violation of the conformation can have a very significant effect on the enzymatic activity or other biological properties of the protein. However, the most important is the primary structure of a protein, which is determined genetically and, in turn, often determines the higher structures of a given protein. Replacement of even one amino acid residue in a polypeptide chain consisting of hundreds of amino acids can significantly change the properties of a given protein and even completely deprive it of biological activity. For example, hemoglobin found in erythrocytes in sickle cell anemia differs from normal hemoglobin A only by replacing the glutamic acid residue in the 6th position of the p-chain with a valine residue, that is, replacing only one of 287 amino acids. However, this replacement is sufficient for the altered hemoglobin to have a sharply impaired solubility and to a significant extent lost its main function of transporting oxygen to tissues. On the other hand, in a strictly defined structure of insulin (Fig. 1), the nature of amino acid residues at the 8th, 9th, and 10th positions of the A chain (between two cysteine ​​residues) does not seem to be significant, since these three residues have a specific specificity; in bovine insulin they are represented by the sequence ala-ser-val, in a sheep - ala-gli-val, in a horse - tre-gli-ile, and in human, pig and whale insulin - tre-ser-ile.

Physicochemical properties

The molecular weight of most proteins ranges from 10-15 thousand to 100 thousand, but there are proteins with a molecular weight of 5-10 thousand and several million. Conventionally, polypeptides with a molecular weight below 5 thousand are referred to as peptides. Most protein fluids and tissues of the body (for example, proteins of blood, eggs, etc.) are soluble in water or in salt solutions. Proteins usually produce opalescent solutions that behave as colloidal. Having many hydrophilic groups in their composition, proteins easily bind water molecules and are in the tissues in a hydrated state, forming solutions or gels. Many proteins are rich in hydrophobic residues and are insoluble in common protein solvents. Such proteins (for example, collagen and elastin of connective tissue, silk fibroin, keratins of hair and nails) are fibrillar in nature, and their molecules are stretched into long fibers. Soluble proteins are usually represented by molecules of a coiled, globular, shape. However, the separation of proteins into globular and fibrillar ones is not absolute, since some proteins (for example, muscle actin) are capable of reversibly converting from a globular configuration to a fibrillar one, depending on environmental conditions.

Like amino acids, proteins are typical amphoteric electrolytes (see Ampholytes), that is, they change their electrical charge depending on the pH of the medium. In an electric field, proteins move to the anode or cathode, depending on the sign of the electric charge of the molecule, which is determined both by the properties of the given protein and the pH of the medium. This movement in an electric field, called electrophoresis, is used for analytical and preparative separation of proteins, usually differing in their electrophoretic mobility. At a certain pH, called the isoelectric point (see), which is not the same for different proteins, the number of positive and negative charges of a molecule is equal to each other, and the molecule as a whole is electrically neutral and does not move in an electric field. This property of the protein is used for their isolation and purification by the method of isoelectric focusing, which consists in the electrophoresis of the protein in a pH gradient created by a system of buffer solutions. In this case, you can select a pH value at which the desired protein precipitates (since the solubility of the protein at the isoelectric point is the lowest), and most of the "contaminating" proteins will remain in solution.

In addition to pH, the solubility of proteins depends significantly on the presence and concentration of salts in solution. High concentrations of salts of monovalent cations (ammonium sulfate is most often used) precipitate most proteins. The mechanism of such precipitation (salting out) consists in the binding of water salts by ions, which forms a hydration shell of protein molecules. Due to dehydration, the solubility of proteins decreases and they precipitate. The same is the mechanism of protein precipitation with alcohols and acetone. The precipitation of proteins by salting out or by organic liquids miscible with water is used to separate and isolate proteins while preserving their natural (native) properties. Under certain precipitation conditions, proteins can be obtained in crystalline form and well purified from other proteins and non-protein impurities. A number of procedures of this kind are used to obtain crystalline preparations of many enzymes or other proteins. Heating protein solutions to high temperatures, as well as protein precipitation with heavy metal salts or concentrated acids, especially trichloroacetic, sulfosalicylic, perchloric, leads to coagulation (clotting) of the protein and the formation of an insoluble precipitate. Under such influences, labile protein molecules denature, lose their biological properties, in particular enzymatic activity, and become insoluble in the original solvent. Denaturation disrupts the native configuration of the protein molecule, and the polypeptide chains form random tangles.

During ultracentrifugation, proteins are deposited in the field of acceleration of centrifugal force at a rate that depends mainly on the size of the protein particles. Accordingly, to determine the molecular weights of proteins, the determination of sedimentation constants in an ultracentrifuge, as well as the rate of protein diffusion, filtering them through molecular sieves, determination of electrophoretic mobility during electrophoresis under special conditions, and some other methods are used.

Methods for the detection and determination of proteins

Qualitative reactions on proteins are based on their physicochemical properties or on the reactions of certain chemical groups in the protein molecule. However, since a large number of various chemical groups are part of a protein molecule, the reactivity of proteins is very high and none of the qualitative reactions to proteins is strictly specific. The conclusion about the presence of a protein can be made only on the basis of a combination of a number of reactions. When analyzing biological fluids, such as urine, where only certain proteins can appear and it is known which substances can interfere with the reaction, even one reaction is enough to establish the presence or absence of proteins. Protein reactions are classified into precipitation and color reactions. The former include precipitation with concentrated acids, and in clinical practice, precipitation with nitric acid is most often used. A typical reaction is also the precipitation of proteins with sulfo-salicylic or trichloroacetic acids (the latter is often used not only to detect proteins, but also to free liquids from proteins). The presence of proteins can also be detected but coagulation by boiling in a weakly acidic medium, precipitation with alcohol, acetone and a number of other reagents. Of color reactions, the biuret reaction is very characteristic (see) - violet coloration with copper ions in an alkaline medium. This reaction depends on the presence of peptide bonds in proteins, which form a colored complex with copper. The name of the biuret reaction comes from the heating product of biuret urea (H 2 N-CO-NH-CO-NH 2), which is the simplest compound that gives this reaction. The xanthoprotein reaction (see) consists in yellow coloration of the protein precipitate when exposed to concentrated nitric acid. Coloring appears due to the formation of nitration products of aromatic amino acids that make up the protein molecule. Millon's reaction gives a bright red color with mercury salts and nitrous acid in an acidic environment. In practice, nitric acid is usually used, which always contains a small impurity of nitrous. The reaction is specific for the phenolic tyrosine radical and therefore is obtained only with proteins containing tyrosine. Adamkevich's reaction is due to the tryptophan radical. It gives a violet color in concentrated sulfuric acid with acetic acid (see Adamkevich's reaction). The reaction is obtained by replacing acetic acid with various aldehydes. When using acetic acid, the reaction is due to glyoxylic acid, which is contained in acetic acid as an impurity. Proteins are usually quantitatively determined by protein nitrogen, that is, by the content of total nitrogen in the protein precipitate washed from low-molecular substances soluble in the precipitator. Nitrogen in biochemical studies and clinical analyzes is usually determined by the Kjeldahl method (see Kjeldahl method). The total protein content in liquids is often determined by colorimetric methods, which are based on various modifications of the biuret reaction. The Lauri method is often used, in which Folin's reagent for tyrosine is used in combination with a biuret reaction (see Lauri method).

Protein classification

Due to the relatively large size of protein molecules, the complexity of their structure and the lack of sufficiently accurate data on the structure of most proteins, there is still no rational chemical classification of proteins. The existing classification is largely arbitrary and is built mainly on the basis of the physicochemical properties of proteins, the sources of their production, biological activity and other, often random, signs. So, according to their physicochemical properties, proteins are divided into fibrillar and globular, hydrophilic (soluble) and hydrophobic (insoluble), etc. According to the source of production, proteins are divided into animal, plant and bacterial; on proteins of muscle, nervous tissue, blood serum, etc.; for biological activity - for enzyme proteins. proteins, hormones, structural. Proteins, contractile proteins, antibodies, etc. It should be borne in mind, however, that due to the imperfection of the classification itself, as well as due to the exceptional diversity of proteins, many of the individual proteins cannot be assigned to any of the groups described here.

All proteins are usually divided into simple, or proteins (actually proteins), and complex, or proteids (complexes of proteins with non-protein compounds). Simple proteins are polymers of amino acids only; complex, in addition to amino acid residues, also contain non-protein, so-called prosthetic groups.

Among simple proteins (proteins), albumin (see), globulins (see) and a number of other proteins are distinguished.

Albumin - readily soluble globular proteins (for example, serum albumin or egg white); dissolve in water and saline solutions with precipitation only when the solution is saturated with ammonium sulfate.

Globulins differ from albumin by insolubility in water and precipitation when the solution is half-saturated with ammonium sulfate. Globulins have a higher molecular weight than albumin and sometimes contain carbohydrate groups.

Proteins also include plant proteins - prolamins (see), which are usually found together with glutelins (see) in cereal seeds (rye, wheat, barley, etc.), forming the bulk of gluten. These proteins are soluble in 70-80% alcohol and insoluble in water; they are rich in proline and glutamic acid residues. Prolamins also include wheat gliadin, corn zein, and barley hordein.

Scleroproteins (proteinond, albumin) - structural proteins, insoluble in water, diluted alkalis, acids and saline solutions. These include fibrillar proteins, mainly of animal origin, which are highly resistant to digestion by digestive enzymes. These proteins are subdivided into connective tissue proteins: collagen (see) and elastin (see); proteins of integuments - hair, nails and hooves, epidermis - keratins (see), which are characterized by a high sulfur content in the form of an amino acid residue - cystine; proteins of cocoons and other secrets of insect silk-secreting glands (for example, cobwebs) - fibroin (see), consisting of more than half of the remains of glycine and alanine.

A special group of proteins are protamines (see) - relatively low molecular weight proteins of a basic nature (in contrast to albumin, globulins and other tissue proteins that have an isoelectric point, usually in a weakly acidic environment). Protamines are found in the semen of some fish and other animals and consist of more than half of diaminomonocarboxylic acids. So, protamines of herring - klupein and salmon - salmin contain about 80% arginine. Other protamines contain, in addition to arginine, lysine or lysine and histidine.

Rice. 2. General scheme of protein biosynthesis. Amino acids (1), interacting with ATP, are activated to form aminoacyladenylates (2); the latter, under the action of the enzyme aminoacyl-tRNA synthetase, combine with transport RNAs, or tRNAs (3), and in the form of an aminoacyl-tRNA complex (4) enter the ribosomes connected to mRNA or polysomes (5). Polysomes are formed by first attaching a small subunit (6) and then a large subunit (7) of ribosomes to mRNA. In the ribosome (8), connected to mRNA, two aminoacyl-tRNAs are attached to the mRNA, as a result of which a peptide bond is formed between them. Thus, the growth of the polypeptide chain (9) occurs, which is released upon completion of its synthesis (10) and is then transformed into a protein (11).

Protein biosynthesis takes place in all cells of living organisms and provides a renewal of body proteins, metabolic processes and their regulation, as well as the growth and differentiation of organs and tissues. Proteins are synthesized in tissues from free amino acids with the participation of nucleic acids (see). The process of protein biosynthesis proceeds with the consumption of energy accumulated in the form of ATP (see. Adenosine phosphoric acids). During the biosynthesis of proteins, the formation of certain proteins of a strictly specific structure is provided, which is encoded in the structural genes (cistrons) of deoxyribonucleic acid, which is found mainly in the chromatin of cell nuclei (see Genetic Code). Information that determines the primary structure of proteins is transmitted to a special type of ribonucleic acids (RNA), called messenger RNA (mRNA), in the form of a complementary nucleotide sequence. This process is called transcription. mRNA combines with ribosomes (see), which are ribonucleoprotein granules, more than half consisting of special ribosomal RNA (rRNA), also synthesized on special cistrons (genes) of DNA. Ribosomes consist of two subparticles, into which they are able to reversibly dissociate with a decrease in the concentration of magnesium ions. Large and small subparticles of ribosomes contain but one RNA molecule with a molecular weight of about 1.7 × 10 6 and 0.7 × 10 6, respectively, and several tens of protein molecules. When combined with ribosomes, mRNA forms polyribosomes, or polysomes, on which the synthesis of polypeptide chains that form the primary structure of proteins occurs. Before joining with ribosomes, amino acids are activated, then they combine with low-polymer RNA carriers, or transport RNAs (tRNAs) in the form of complexes, with which they enter the ribosomes. The general scheme of protein biosynthesis is shown in Fig. 2.

The activation of amino acids occurs when they interact with ATP with the formation of aminoacyladenylate and the release of pyrophosphate: amino acid + ATP = aminoacyladenylate + pyrophosphate. Aminoacyladenylate is a mixed anhydride formed by a phosphoric acid residue of adenosine monophosphate and a carboxyl group of an amino acid, and is an activated form of an amino acid. From aminoacyladenylate, the amino acid residue is transferred to tRNA, specific for each amino acid, and enters the ribosomes in the form of aminoacyl-tRNA. The formation of aminoacyladenylate and the transfer of an amino acid residue to tRNA are catalyzed by the same enzyme (aminoacyladenylate synthetase, or aminoacyl-tRNA synthetase), which is strictly specific for each amino acid and each tRNA. All tRNAs have a relatively low molecular weight (about 25,000) and contain about 80 nucleotides. They have a cruciform configuration, reminiscent of a clover leaf, and the nucleotide chain forms a double-stranded structure held by complementary bases and becomes single-stranded only in the region of the loops. The beginning of the nucleotide chain, usually represented by a 5'-guanyl nucleotide, is located near the terminal, often exchanging group of two cytidylic acid and adenosine residues with a free 3'-OH group, to which the amino acid residue is attached. On the loop, located at the opposite end of the tRNA molecule, there is a base triplet, complementary to the triplet encoding the given amino acid (codon), and called the anticodon. The nucleotide sequence of many tRNAs has already been established, and their complete structure is known.

The determined amino acid sequence in the primary structure of the synthesized polypeptide chain is provided by information recorded in the mRNA nucleotide sequence, reflecting the corresponding sequence in DNA cistrons. Each amino acid is encoded by specific triplets of mRNA nucleotides. These triplets (codons) are presented in table. 2. Their decoding made it possible to establish the RNA nucleotide code, or amino acid code, that is, the method by which the translation occurs, or the translation of information recorded in the RNA nucleotide sequence into the primary structure of proteins, or the sequence of amino acid residues in the polypeptide chain.

Table 2. RNA-AMINO ACID CODE

Note: U - uridylic acid, C - cytidylic acid, A - adenylic acid, G - guanylic acid. Three letters indicate the corresponding amino acid residue: eg. Phen - phenylalanine. Ile - isoleucine, Glu - glutamic acid, Gln - glutamine, etc. Triplets UAA, UAG, UGA do not encode amino acids, but determine the termination of the polypeptide chain.

As can be seen from the table, out of 64 possible triplets (61 encode certain amino acids, that is, they are “semantic.” Three triplets - UDD, UAG and UGA - do not encode amino acids, but their role is to complete (terminate) the synthesis of the growing polypeptide chain. is degenerate, that is, almost all amino acids are encoded by more than one triplet of nucleotides.Thus, 3 amino acids - leucine, arginine and serine - are encoded by six codons, 2 - methionine and tryptophan - have only one codon, and the remaining 15 - from 2 to 4 The translation process is carried out with the help of tRNA loaded with amino acids. Aminoacyl-tRNA is attached by its complementary triplet (anticodon) to the mRNA codon in the ribosome. Another aminoacyl-tRNA is attached to the adjacent mRNA codon. The first tRNA attaches its amino acid residue to the second carboxyl group. amino acids, with the formation of a dipeptide, and itself is freed and separated from the ribosome. Further, as p ibosomes but mRNA strands from the 5 "end to the 3" end, the third aminoacyl RNA is attached; there is a connection of the dipeptide with the carboxyl end of the amino group of the third amino acid with the formation of a tripeptide and the release of the second tRNA, and so on until the ribosome passes the entire region encoding this protein on the mRNA corresponding to the cistron of DNA. Then the termination of protein synthesis occurs, and the resulting polypeptide is freed from the ribosome. The first ribosome in the polysome is followed by the second, third, etc., which sequentially read information on the same mRNA strand in the polysome. Thus, the growth of the polypeptide chain occurs from the N-terminus to the carboxyl (C-) terminus. If you suppress protein synthesis, for example, using the antibiotic puromycin, you can get unfinished polypeptide chains with an incomplete C-terminus at different stages. Aminoacyl-tRNA is first attached to a small ribosomal subunit, and then transferred to a large subunit, on which the polypeptide chain grows. According to the hypothesis of A.S.Spirin, during the work of the ribosome during the biosynthesis of proteins, there is a repeated closing and opening of ribosome subparticles. To reproduce protein synthesis outside the body, in addition to ribosomes, mRNA, and aminoacyl-tRNA, the presence of guanosine triphosphate (GTP) is necessary, which is cleaved to HDP and regenerated again during the growth of the polypeptide chain. It also requires the presence of several protein factors that seem to play an enzymatic role. These so-called transfer factors interact with each other and require the presence of sulfhydryl groups and magnesium ions for their activity. In addition to translation itself (that is, the growth of a polypeptide chain in a specific sequence corresponding to the structural DNA gene and the transmitted nucleotide sequence in mRNA), the beginning (or initiation) of translation and its completion (or termination) play a special role. The initiation of protein synthesis in the ribosome, at least in bacteria, begins with special codons - initiators in mRNA - AUG and GUG. First, a small subunit of the ribosome binds to such a codon, then formylmethionyl-tRNA is attached to it, with which the synthesis of the polypeptide chain begins. Due to the special properties of this aminoacyl-tRNA, it can be transferred to a large subunit like peptidyl-tRNA, and thus begin the growth of the polypeptide chain. For initiation, GTP and protein initiation factors are required (three are known). Termination of the growth of the polypeptide chain occurs on the “meaningless” codons of the UAA, UAH, or UGA. Apparently, these codons bind to a special protein termination factor, which, in the presence of another factor, promotes the release of the polypeptide.

The components of the protein biosynthesis system are synthesized mainly in the cell nucleus. All types of RNA are synthesized on the DNA matrix during transcription. involved: in this process: rRNA, mRNA and tRNA. So, rRNA and mRNA are synthesized in the form of very large molecules, and even in the cell nucleus they undergo a process of "maturation", during which a part (very significant for mRNA) of molecules is cleaved and disintegrated without leaving the cytoplasm, and functioning molecules that are part of the original synthesized, enter the cytoplasm to the sites of protein synthesis. Before getting into the composition of the polysome, mRNA, apparently, from the moment of synthesis, binds to special protein particles, "informofers", and in the form of a ribonucleoprotein complex is transferred to the ribosomes. Ribosomes, apparently, also "mature" in the cytoplasm, some of the proteins are attached to the precursors of ribosomes emerging from the nucleus, already in the cytoplasm. It should be noted that lower, non-nuclear organisms (prokaryotes), which include bacteria, blue-green algae and viruses, have some differences from higher organisms in the components of the protein biosynthesis system, and especially in its regulation. Ribosomes in prokaryotes are somewhat smaller in size and differ in composition; the process of transcription and translation is directly linked into one whole. At the same time, in higher nuclear organisms (eukaryotes), RNA is also formed in cytoplasmic organelles, mitochondria and chloroplasts (in plants), which have their own protein synthesis system and their own genetic information in the form of DNA. In terms of its structure, the system of protein synthesis in mitochondria and chloroplasts is similar to that in prokaryotes and differs significantly from the system found in the nucleus and cytoplasm of higher animals and plants.

The regulation of protein biosynthesis is a very complex system and allows the cell to quickly and clearly respond to changes in the environment surrounding the cell by stopping or inducing the synthesis of various proteins, often with enzymatic activity. In bacteria, the suppression of protein synthesis is carried out mainly with the help of special proteins - repressors (see Operon), synthesized by special regulator genes. The interaction of a repressor with a metabolite coming from the environment or synthesized in the cell can suppress or, conversely, activate it, thus regulating the synthesis of one protein or several interrelated proteins, especially enzymes that are also synthesized interconnectedly on one operon. In higher organisms, in the process of differentiation, tissues lose the ability to synthesize a number of proteins and specialize in the synthesis of a smaller number of proteins necessary for the function of this tissue, for example, muscles. Such blocking of the synthesis of a number of proteins occurs, apparently, at the level of the genome (see) with the help of nuclear proteins - histones (see) that bind non-functional DNA regions. However, during regeneration, malignant growth, and other processes associated with dedifferentiation, such blocked sites can be derepressed and supply mRNA for the synthesis of proteins unusual for a given tissue. Nevertheless, in higher organisms, there is a regulation of protein synthesis in response to certain stimuli. Thus, the action of a number of hormones is to induce protein synthesis in the tissue that is the "target" of this hormone. Such induction, apparently, occurs by the binding of the hormone to a specific protein of the given tissue and the activation of the gene through the formed complex.

The process of protein biosynthesis and its regulation require extreme clarity, precision and coherence in the work of all components of the system. Even small violations of this accuracy lead to a violation of the primary structure of proteins and severe pathological consequences. Genetic disorders, for example, replacement or loss of one nucleotide in a structural gene, lead to the synthesis of an altered protein, often devoid of biological activity. Such changes underlie congenital metabolic disorders, which, in essence, include all hereditary diseases (see). On the other hand, a number of proteins and enzymes can differ not only in different biological species, but also in different individuals, while maintaining their biological activity. Often, these proteins have different immunological and electrophoretic properties. In human populations, many examples of the so-called protein polymorphism are described, when in different individuals, and sometimes in the same individual, two or more dissimilar proteins can be found that have the same function, such as, for example, hemoglobin (see), haptoglobin (see) and some others.

Proteins in the diet

Of the many nutrients, proteins play the most important role. They are sources of essential amino acids and the so-called nonspecific nitrogen necessary for the synthesis of proteins in the human body. Severe protein deficiency in the diet leads to severe dysfunctions of the body (see Alimentary dystrophy). The state of health, physical development and working capacity of a person largely depends on the level of protein supply, and in young children, to a certain extent, mental development. If we take into account all plant and animal proteins produced for nutrition, then on average, about 58 g per day will be needed for each inhabitant of the Earth. In fact, more than half of the population, especially in developing countries, does not get this amount of protein. The global deficit of dietary protein should be classified as one of the most acute economic and social problems of our time (see Protein Crisis). As such, establishing optimal protein levels in diets is of paramount importance.

Proteins are required in greatest quantities during periods of intense growth. However, in an organism that has reached maturity, vital processes are associated with a continuous waste of protein substances and, consequently, the need to replenish these losses with food. In accordance with the recommendations of the FAO / WHO Expert Group, the calculation of the need for protein nitrogen should be carried out according to the formula: R = 1.1 (U b + F b + S + G), where R is the need for protein nitrogen; U b - excretion of nitrogen in the urine; F b - excretion of nitrogen with feces; S - loss of nitrogen due to desquamation of the epidermis, growth of hair, nails, release of nitrogen with sweat during non-intense sweating; G - nitrogen retention during growth (calculated per 1 kg of mass per day).

The coefficient 1.1 reflects the additional expenditure of proteins (on average 10%) arising from stress reactions and adverse effects on the body. The limits of individual variations in protein requirements are taken equal to ± 20%. The official recommendations of the FAO / WHO expert group are reflected in table. 3.

Table 3. AVERAGE DAILY PROTEIN REQUIREMENT (subject to its complete assimilation) *

• The nitrogen demand is multiplied by a factor of 6.25.

Obviously, the given values ​​but correspond to the optimal supply of proteins to a person and should be attributed to the minimum level of their content in the diet, if not observed, a relatively rapid development of serious consequences of protein deficiency is inevitable. The actual consumption of proteins in most economically developed countries is 1.5 and even 2 times higher than the above figures. According to the concept of a balanced diet, the optimal human need for proteins depends on many factors, including the physiological characteristics of the body, the qualitative characteristics of food proteins and the content of other nutrients in the diet.

In the USSR, the values ​​of the population's needs for proteins are recorded in the physiological nutritional norms officially approved by the Ministry of Health, which are periodically reviewed and updated. Physiological nutritional norms are average indicative values ​​reflecting the optimal needs of certain groups of the population in basic nutrients and energy (Table 4).

They provide for the differentiation of protein requirements, depending on gender, age, nature of work, etc. The recommended values ​​are calculated on the basis of studying the characteristics of protein metabolism and nitrogen balance in the corresponding population groups, and they are significantly higher than the minimum protein requirements required for maintaining nitrogen balance. An excess of proteins is necessary to ensure additional expenditures of the body associated with physical and nervous stress, adverse environmental influences, as well as to maintain an optimal immunological status. The consumption rates of the most valuable proteins of animal origin have been specially highlighted in the norms.

Physiological nutritional norms are the basis for planning the production of certain food products. When assessing the usefulness of individual protein products, their amino acid composition, the degree of digestibility by enzymes of the digestive tract and integral indices of digestibility established as a result of biological experiments are taken into account. In practice, with a certain degree of convention, protein products are divided into two groups. The first includes products of animal origin: milk, meat, eggs, fish, the proteins of which are easily and completely absorbed by the human body; to the second - most of the products of plant origin, in particular wheat, rice, corn and other cereals, the proteins of which are not fully assimilated by the body. The conventionality of such a division is emphasized by the high biological value of a number of plant proteins (potatoes, buckwheat, soybeans, sunflower) and the low biological value of the proteins of some animal products (gelatin, skin, tendons, etc.). The reasons for the low digestibility of fibrillar proteins (keratin, elastin and collagens) lie in the peculiarities of their tertiary structure and the difficulty of digestion by enzymes of the digestive tract. On the other hand, the assimilation of a number of proteins of plant origin may depend on the structure of plant cells and the difficulties encountered in contacting proteins with digestive enzymes.

The completeness of the use of individual proteins by a person or their biological value is primarily determined by the degree to which their amino acid composition corresponds to the differentiated needs of the body and, to some extent, the amino acid composition of the body. The huge variety of naturally occurring proteins is mainly composed of 20 amino acids, 8 of them (tryptophan, leucine, isoleucine, valine, threonine, lysine, methionine and phenylalanine) are indispensable for humans, since they cannot be synthesized in body tissues (see Amino acids ). For young children, the ninth essential amino acid is histidine. The rest of the amino acids are nonessential and can be regarded in the diet mainly as suppliers of nonspecific nitrogen. It has been established that the best assimilation of food proteins is achieved by balancing its amino acid composition with “ideal” amino acid scales. As such a scale, the so-called FAO Provisional Amino Acid Scale was proposed in 1957. It was later proved that the content of a number of amino acids in it, especially tryptophan and methionine, was not determined quite accurately. In accordance with the results of biological studies, scales of the amino acid composition of proteins of chicken eggs and human milk have been recommended as optimal in recent years. The proteins of these two products are by nature intended for the nutrition of developing organisms and are almost completely utilized both in experiments on experimental animals and when used in the nutrition of young children.

To determine the correspondence of the amino acid composition of proteins to human needs, a number of indices have been proposed, each of which has only limited value. Among them, mention should be made of the H / O index, which reflects the ratio of the sum of essential amino acids (H in mg) to the total nitrogen content of proteins (O in g), which helps to determine the ratio of the nitrogen of essential, or essential, amino acids and non-specific nitrogen. The lower the H / O value, the higher the nonspecific nitrogen content. For proteins of milk and eggs, this index is relatively high - 3.1-3.25, for meat - 2.79-2.94; for wheat - 2. Great importance is attached to the indicator of amino acid scor, which makes it possible to obtain a more complete judgment about the biological value of a protein on the basis of its chemical. composition.

The scor method is based on calculating the percentage of each of the essential amino acids in the test product in comparison with the ideal amino acid scales.

For this purpose, for each of the essential amino acids of the protein under study, the value of I research is calculated, equal to A research / H research, reflecting the ratio of each essential amino acid (A in mg) to the sum of essential amino acids (H in g); the resulting figure is compared with the value of I st, equal to A st / H st for the same amino acid, calculated on a standard scale. As a result of dividing the Iresl values ​​by Ist and multiplying by 100, the amino acid score for each of the essential amino acids is obtained. The limiting biological value of the protein under study is the amino acid, the amino acid rate for which is the smallest. Along with the preliminary FAO scale, the amino acid scales of chicken eggs and human milk are used as standard scales (Table 5).

Table 5. STANDARD AMINO ACID SCALES

In accordance with the indicators of amino acid scor (Table 6), proteins of a number of cereals, especially wheat (50%; limiting amino acids - lysine and threonine) have the lowest biological value; corn (45%; limiting amino acids - lysine and tryptophan); millet (60%; limiting amino acids - lysine and threonine); peas (60%; limiting amino acids - methionine and cystine). The indicator of the amino acid rate of the limiting amino acid sets the limit for the use of nitrogen of a given type of protein for plastic purposes. An excess of other amino acids contained in protein can only be used as a source of non-specific nitrogen or for energy needs of the body. The method of studying the amino acid composition is one of the main methods for assessing the quality of proteins. Typically, it provides digestibility indicators that are close to those of longer and more costly methods for biological determination of protein value. At the same time, the establishment in a number of cases of reliable discrepancies between the indicated indicators makes it necessary to resort to the integral methods of biol in the study of new protein products. assessments both in laboratory animals and directly in humans. These methods are based on the study in balance experiments of the completeness of the use of individual proteins by growing animals (indicator of protein efficiency of the diet), the ratio of nitrogen retained by the body to nitrogen adsorbed from the intestine (indicator of biological value), the ratio of adsorbed nitrogen to total nitrogen of food (indicator of true digestibility) etc. When setting up studies on the study of biol, the value of protein, it is imperative to provide a sufficiently high-calorie diet, balance it for all essential nutritional factors (see Balanced nutrition) and a relatively low level of proteins - within 8-10% of the total calorie content ( see Metabolism and Energy). Comparison of indicators of amino acid scor and protein utilization, determined in experiments on experimental animals for some products, is presented in table. 6.

Table 6. COMPARISON OF AMINO ACID RATE AND PROTEIN UTILIZATION

An important advantage of biological methods for evaluating proteins is their integrity, which makes it possible to take into account the entire complex of properties of products that affect the assimilation of proteins included in them. When studying the biological value of individual proteins, one should not forget that almost all diets use not individual proteins, but their complexes, and, as a rule, various proteins complement each other, providing some average indicators of protein nitrogen assimilation. With a variety of mixed diets, the protein digestibility rate is relatively constant and approaches 85%, which is often used in practical calculations.

Rice. 2. Danielle's reaction to proteins containing tyrosine, tryptophan, histidine in the auricle of the heart.

Histochemical methods for the detection of proteins are, as a rule, based on biochemical methods adapted for the determination of proteins in thin tissue sections. It should be borne in mind that a biochemical reaction can be used as a histochemical reaction if the reaction product has a stable color color, precipitates and does not have a pronounced tendency to diffusion. Histochemical methods for detecting proteins in tissues are based on the identification of certain amino acids that make up proteins (for example, Millon's reaction to tyrosine, Sakagushi's reaction to arginine, Adams's reaction to tryptophan, tetrazonium combination reaction to histidine, tyrosine, tryptophan, etc.), on the identification of certain chemical groups (NH 2 =, COOH -, SH =, SS =, etc.), on the use of some physical and chemical methods (color. Fig. 1-3), determination of the isoelectric point, etc. Finally, the presence of certain amino acids in a tissue section can be determined indirectly by determining the presence in tissues of enzymes associated with these amino acids (for example, D-amino acid oxidase). Some simple proteins (collagen, elastin, reticulin, fibrin) are detected in sections using numerous histological methods, among which the so-called polychromic methods are preferable (Mallory's method and its modifications, Romeis's orsein pyrofuchsin method, etc. Proteins are also detected using luminescence microscopy methods The localization of proteins in tissues (myosins, albumins, globulins, fibrin, etc.) can be obtained using the method of labeled antibodies according to Koons, etc. These methods and their modifications allow to quite accurately identify and determine the localization of individual proteins that differ from each other the content of certain amino acids. Methods for the quantitative determination of proteins are being developed, for example, a method for the determination of proteins by the indirect reaction of labeled antibodies, as well as the determination of SH-groups by the method of Barnett and Seligman (see Amino acids, histochemical methods for the detection of amino acids). All the above methods for the detection of proteins in tissues have q residual specificity and give quite reliable results. Fixation of tissue material using these methods is different. In most cases, the most suitable fixatives should be considered ethyl or methyl alcohol, anhydrous acetone, a mixture of ethyl alcohol with formalin, a solution of trichloroacetic acid on alcohol, in some cases (for proteids of the anterior pituitary gland) formalin is used. The choice of fixator depends on the method, the fixation time depends on the total amount and nature of the tissue. Cryostat or paraffin sections can be used.

Radioactive proteins

Radioactive proteins are protein substances, the molecule of which contains one or more atoms of radioactive isotopes of any elements. With radioactive labeling of proteins, it is necessary to ensure the strength and the greatest possible preservation of the protein molecule. The isotopes 3 H and 14 C are mainly used as a radioactive label of proteins for biochemical experimental studies; when producing radiopharmaceuticals based on proteins, iodine isotopes 125 I and 131 I are used, as well as isotopes 111 In, 113m In, 99m Tc, etc. peptide. The labeled protein is purified from unbound iodide and other impurities (by gel filtration, dialysis, adsorption, ion exchange, isoelectric precipitation, etc.). If the proteins do not contain tyrosine, for iodization, substituents containing radioactive iodine are introduced into it, or tyrosine-containing analogs are used, or they resort to a label with other radioactive isotopes (see).

Radioactive proteins are important in the study of catabolism and metabolism of protein substances in experimental biochemical studies. In addition, they are used in in vivo and in vitro radioisotope diagnostics when studying the functional state of many organs and body systems in the case of various diseases. In in vivo studies, human serum albumin labeled with radioactive isotopes of iodine (125 I and 131 I), as well as micro- and macro-albumin aggregates obtained on its basis by thermal denaturation and aggregation with the same label, finds the greatest application in in vivo studies. With the help of labeled albumin, hemodynamic and regional blood circulation parameters, the volume of circulating blood and plasma can be determined, the heart and large vessels are scanned (see Scanning), as well as brain tumors. Microaggregates of albumin are used for scanning the liver and stomach, determining liver blood flow, and macroaggregates for scanning the lungs.

Radioactive proteins have found wide application in the determination of trace amounts of hormones, enzymes and other protein substances in tissues and environments of the body of animals and humans in in vitro studies.

Bibliography: Proteins, ed. G. Neurath and K. Bailey, trans. from English, t. 1-3, M., 1956 -1959, bibliogr .; Biosynthesis of Protein and Nucleic Acids, ed. A.S. Spirina, M., 1965; F. Gaurovnz Chemistry and functions of proteins, trans. from English .. M., 1965; Ichas M. Biological code, trans. from English, M., 1971; Kiselev L. L. et al. Molecular bases of protein biosynthesis. M., 1971; Poglaov BF Structure and functions of contractile proteins, M., 1965; Spirin A.S. and Gavrilova L.P. Ribosoma, M., 1971; Chemistry and Biochemistry of Nucleic Acids, ed. I. B. Zbarsky and S. S. Debova, L., 1968; Advances in protein chemistry, ed. by M. L. Anson a. J. T. Edsall, v. 1-28, N. Y. 1944-1974; Hess G. P. a. R upley J. A. Structure and function of proteins, Ann. Rev. Biochcm., V. 40, p. 1013, 1971; In vitro procedures with radioisotopes in mcdlcinc, Proceedings of the symposium, Vienna, 1970; M ar gl (n A. a. Nerrif ield R.B. Chemical synthesis of peptides and proteins, Ann. Rev. Biochem., V. 39, p. 841, 1970; Proteins, composition, structure, and function, ed. By H. Neurath, v. 1-5, NY-L., 1963-1970.

B. in nutrition- Lavrov B. A. Textbook of nutritional physiology, p. 92, M., 1935; Molchanova OP The value of protein in nutrition for a growing and adult organism, in the book: Vopr. p., ed. O. P. Molchanova, V. 2, p. 5, M., 1950; P about to r ovsky A. A. On the question of the needs of various groups of the population in energy and basic nutrients, Vestn. USSR Academy of Medical Sciences, no. 10, p. 3, 1966, bibliogr .; he, Fieiologo-biochemical bases of development of baby food products, M., 1972; Energy

Histochemical methods of B.'s detection in tissues- Kiseli D. Practical microtechnics and histochemistry, trans. with fan., with. 119, 152, Budapest "1962; L and l-l I r. Histopathological technique and Factual histochemistry, trans. from English, p. 509, M., 1969; P and r with E. Histochemistry, trans. e English .. M., 1962; Principles and methods of gn-rgo-cytochemical analysis in pathology, ed. A.P. Avtsyna and others, p. 238, JI., ". 971; Peagse A. G. E. Histochemistry, Vol. 1-2, Edinburgh-L., 1969-1972.

I. B. Zbarsky; A. A. Pokrovsky (pit.), V. V. Sedov (glad.), R. A. Simakova (hist.).

Proteins are one of the most important and vital substances in the human body.

How often the deficiency of vitamins and minerals we almost every year feel on the piled blues and fatigue and willingly, out of habit, blame it on "vitamin deficiency." But it's important to understand that many health problems can be associated with a quality protein deficiency. And this, unfortunately, we are very rarely worried about.

How can we determine if our body has enough protein and is it time to replenish its reserves? Protein deficiency in the body can be seen by the following signs:

Pulls for sweet

This is one of the main signs of a lack of protein when you pounce on sweets and the feeling of hunger does not leave you. It just so happens that while limiting protein foods, we are in no hurry to lean on meat and eggs - the main task of proteins is to keep blood sugar levels. And it is sweets that help to quickly correct the situation.

Poor concentration

The concentration will only be excellent when the blood sugar level is balanced. And when this level is subject to constant fluctuations, then a feeling of clouding of consciousness may well arise, in which it is impossible to concentrate on work or study. Therefore, remember: the brain must be constantly fed with proteins.

Hair loss
It is important to know that proteins are an irreplaceable building material for all cells, including hair follicles. When these follicles are strong, the hair will be held on the head, but with a chronic lack of proteins, they begin to actively fall out.

Weakness

It is generally known that proteins are also the main building material for muscles. Therefore, when there is a lack of protein in the body, the muscles begin to decrease in size. Over time, this condition can lead to chronic weakness and loss of strength.

Soreness
The entire immune system of a person directly depends on the systematic influx of protein. That is why fairly frequent colds and infectious diseases are a clear indication of a lack of proteins.

What is the protein

Animal and vegetable proteins

Most plant foods contain no less protein than milk or chicken. But the human body is designed in such a way that, as usual, the protein is partially absorbed, everything else is excreted in the urine. You should eat protein from both plant and animal sources - but this is ideal. If you're on a vegetarian diet, you just need to balance your diet to compensate for the lack of animal protein.

Animal protein

Which foods contain animal protein:

• kefir;
• hard cheeses;
• seafood and fish;
• cottage cheese;
• milk;
• egg white;
• dietary meat - rabbit and turkey;
• red meat;
• hen.

All of these foods contain both protein and fat, but not in the least amount. It should not be forgotten that among products containing protein, it is recommended to give preference to dairy products, the fat content of which is no more than 3%, skinless chicken and lean meat. As for cheeses, then the fat content is allowed up to 40%.

Vegetable protein

Since vegetarianism is in vogue at the moment, we will tell you which plants contain a large amount of protein.

So nuts:

• Brazilian nut;
• macadamia nut;
• hazelnut;
• pine nuts;
• walnuts;
• almond oil and almonds.

Vegetable protein is digestible from cereals, however, you need to know, at least for combining with animal protein, which cereals contain protein in large quantities:

• peanut;
• quinwa;
• oats;
• pearl barley;
• peas;
• lentils;
• buckwheat.

The most favorable combination is vegetable and animal protein at the same time on one plate. And for this reason, we advise you to combine dairy products, fish and meat with vegetable protein, for example, with vegetables.

"Modification of leaves" - Venus flytrap. Cereus. Remember:? What are the functions of the sheet? ? Thorns. b) Antennae. Nepentes. Trichocerus. Mamillaria. Mouse peas. The Venus flytrap is an insectivorous plant. Leaf modifications: a) Spines. Tendril. Peas. c) Insectivorous leaves. Modifications of leaves. Barberry. For what purpose are the organs being modified?

"Organs of a flowering plant" - Organs of a flowering plant. The shoot is a part of the stem with leaves and buds located on it. The leaf is one of the main organs of the plant, occupying a lateral position in the shoot. Herbaceous Stem Woody. Part of the escape. Root Shoot Stem Leaf Flower Fruit Seed. Reproductive organ of flowering plants.

"Fruit" - Presentation Topic. Dry fruits. Classification of fruits. Plan. People. Dry Juices Rozkrivnі Nerozkrivnі 2. For a few days. Budova to the fruit Fertile Nasinnya. Hot Pod. Self-width. 1. Fruit growth. 2. Expanded fruit. 3. Value. Fruit. Yabluko Orange. Berry. Expanded fruit. Box Cim'yanka.

"Plant tissue" - Morphology and anatomy. Companion cells. 20. Trachea. 16. Cuticle and waxy bloom are found on fruits, leaves, stems, parts of a flower. Mechanical fabrics. Companion cells are located next to each segment. Parenchyma. The stomata are more often located on the underside of the leaf. Conductive fabrics. The homogeneous body of lower plants is called thallus, or thallus.

"Life forms of plants" - Systematics introduced in 1735. Systematics according to the ecological (biogeocenotic) character. Systematics of K. Linnaeus. Sylvanas Petrophytes. Caudex is developed in elecampane tall (Elena), wormwood. Protants. Geophytes. Binary nomenclature, i.e. genus name, species name. Stepants Polyudants. The appearance of a plant, formed as a result of adaptation to environmental conditions.

"Plants and their fruits" - Shrubs. Wood. There are also special cases of gigantism of grasses in the Sayan Mountains (Krasnoyarsk Territory). The height of the grasses is from a few millimeters to several meters. Cereals. In the Sikhote-Alin mountains, the grass often reaches a height of 3-3.5 m. According to botanists, all fruits containing seeds are fruits. Fruit and berry bushes are of great economic importance: currants, gooseberries, etc.

There are 13 presentations in total

# 1. Proteins: peptide bond, their detection.

Proteins are macromolecules of linear polyamides formed by a-amino acids as a result of the polycondensation reaction in biological objects.

Protein Are high molecular weight compounds built from amino acids... 20 amino acids are involved in the creation of proteins. They bind to each other in long chains that form the backbone of a high molecular weight protein molecule.

Functions of proteins in the body

The combination of the unique chemical and physical properties of proteins provides precisely this class of organic compounds with a central role in the phenomena of life.

Proteins have the following biological properties, or perform the following main functions in living organisms:

1. Catalytic function of proteins. All biological catalysts - enzymes are proteins. Thousands of enzymes have now been characterized, many of them isolated in crystalline form. Almost all enzymes are powerful catalysts that increase reaction rates by at least a million times. This function of proteins is unique, not found in other polymer molecules.

2. Nutritional (reserve function of proteins). These are, first of all, proteins intended for nutrition of the developing embryo: milk casein, egg ovalbumin, storage proteins of plant seeds. A number of other proteins are undoubtedly used in the body as a source of amino acids, which, in turn, are precursors of biologically active substances that regulate the metabolic process.

3. Transport function of proteins. The transport of many small molecules and ions is carried out by specific proteins. For example, the respiratory function of blood, namely the transfer of oxygen, is performed by molecules of hemoglobin, a protein of red blood cells. Serum albumin is involved in lipid transport. A number of other whey proteins form complexes with fats, copper, iron, thyroxine, vitamin A and other compounds, ensuring their delivery to the appropriate organs.

4. Protective function of proteins. The main function of protection is performed by the immunological system, which ensures the synthesis of specific protective proteins - antibodies - in response to the entry of bacteria, toxins or viruses (antigens) into the body. Antibodies bind antigens, interacting with them, and thereby neutralize their biological effect and maintain the normal state of the body. Clotting of a protein in blood plasma - fibrinogen - and the formation of a blood clot, which prevents blood loss in case of injury, is another example of the protective function of proteins.

5. The contractile function of proteins. Many proteins are involved in the act of muscle contraction and relaxation. Actin and myosin, specific proteins of muscle tissue, play the main role in these processes. The contractile function is also inherent in the proteins of subcellular structures, which ensures the subtlest processes of cell life,

6. Structural function of proteins. Proteins with this function rank first among other proteins of the human body. Structural proteins such as collagen in connective tissue are widespread; keratin in hair, nails, skin; elastin - in the vascular walls, etc.

7. Hormonal (regulatory) function of proteins. The body's metabolism is regulated by a variety of mechanisms. In this regulation, an important place is occupied by hormones produced by the endocrine glands. A number of hormones are represented by proteins, or polypeptides, for example, hormones of the pituitary gland, pancreas, etc.

Peptide bond

Formally, the formation of a protein macromolecule can be represented as a polycondensation reaction of α-amino acids.

From a chemical point of view, proteins are high-molecular nitrogen-containing organic compounds (polyamides), the molecules of which are built from amino acid residues. Monomers of proteins are α-amino acids, the common feature of which is the presence of a carboxyl group -COOH and an amino group -NH 2 at the second carbon atom (α-carbon atom):

Based on the results of studying the products of protein hydrolysis and put forward by A.Ya. Danilevsky ideas about the role of peptide bonds -CO-NH- in the construction of a protein molecule, the German scientist E. Fischer proposed a peptide theory of the structure of proteins at the beginning of the 20th century. According to this theory, proteins are linear polymers of α-amino acids linked by a peptide linkage - polypeptides:

In each peptide, one terminal amino acid residue has a free α-amino group (N-terminus), and the other has a free α-carboxyl group (C-terminus). It is customary to depict the structure of peptides starting from the N-terminal amino acid. In this case, amino acid residues are indicated by symbols. For example: Ala-Tyr-Leu-Ser-Tyr- - Cys. This entry designates a peptide in which the N-terminal α-amino acid is ­ is alanine, and the C-terminal - cysteine. When reading such a record, the endings of the names of all acids, except for the latter, change to - "silt": alanyl-tyrosyl-leucyl-seryl-tyrosyl-β-cysteine. The length of the peptide chain in peptides and proteins found in the body ranges from two to hundreds and thousands of amino acid residues.

No. 2. Classification of simple proteins.

TO simple (proteins) include proteins that give only amino acids during hydrolysis.

Proteinoids ____ simple proteins of animal origin, insoluble in water, salt solutions, dilute acids and alkalis. Mainly supporting functions (e.g. Collagen, keratin

protamine - positively charged nuclear proteins, with a molecular weight of 10-12 kDa. About 80% are composed of alkaline amino acids, which gives them the ability to interact with nucleic acids through ionic bonds. They take part in the regulation of gene activity. Well soluble in water;

histones - nuclear proteins that play an important role in the regulation of gene activity. They are found in all eukaryotic cells, and are divided into 5 classes, differing in molecular weight and amino acid. The molecular weight of histones is in the range from 11 to 22 kDa, and the differences in the amino acid composition concern lysine and arginine, the content of which varies from 11 to 29% and from 2 to 14%, respectively;

prolamins - insoluble in water, but soluble in 70% alcohol, features of the chemical structure - a lot of proline, glutamic acid, no lysine ,

glutelins - soluble in alkaline solutions ,

globulins - proteins insoluble in water and in a semi-saturated solution of ammonium sulfate, but soluble in aqueous solutions of salts, alkalis and acids. Molecular weight - 90-100 kDa;

albumin - proteins of animal and plant tissues, we will dissolve in water and saline solutions. The molecular weight is 69 kDa;

scleroproteins - proteins of the supporting tissues of animals

Silk fibroin, egg serum albumin, pepsin, etc. are examples of simple proteins.

No. 3. Methods for isolation and precipitation (purification) of proteins.

No. 4. Proteins as polyelectrolytes. Isoelectric point of protein.

Proteins are amphoteric polyelectrolytes, i.e. exhibit both acidic and basic properties. This is due to the presence in protein molecules of amino acid radicals capable of ionization, as well as free α-amino and α-carboxyl groups at the ends of peptide chains. Acidic properties of the protein are given by acidic amino acids (aspartic, glutamic), and alkaline properties - by basic amino acids (lysine, arginine, histidine).

The charge of a protein molecule depends on the ionization of acidic and basic groups of amino acid radicals. Depending on the ratio of negative and positive groups, the protein molecule as a whole acquires a total positive or negative charge. When acidifying the protein solution, the degree of ionization of the anionic groups decreases, and the cationic ones increase; when alkalizing, the opposite is true. At a certain pH value, the number of positively and negatively charged groups becomes the same, an isoelectric state of the protein appears (the total charge is 0). The pH value at which a protein is in the isoelectric state is called the isoelectric point and is denoted pI, similar to amino acids. For most proteins, pI lies in the range of 5.5-7.0, which indicates a certain predominance of acidic amino acids in proteins. However, there are also alkaline proteins, for example, salmin - the main protein from salmon milk (pl = 12). In addition, there are proteins in which pI has a very low value, for example, pepsin, an enzyme of gastric juice (pl = l). At the isoelectric point, proteins are very unstable and easily precipitate, having the least solubility.

If a protein is not in an isoelectric state, then in an electric field its molecules will move to the cathode or anode, depending on the sign of the total charge and at a rate proportional to its value; this is the essence of the electrophoresis method. This method can separate proteins with different pI values.

Although proteins have buffer properties, their capacity at physiological pH values ​​is limited. The exception is proteins containing a lot of histidine, since only the histidine radical has buffering properties in the pH range 6-8. There are very few such proteins. For example, hemoglobin, containing almost 8% histidine, is a potent intracellular buffer in erythrocytes, maintaining blood pH at a constant level.

No. 5. Physicochemical properties of proteins.

Proteins have different chemical, physical and biological properties, which are determined by the amino acid composition and spatial organization of each protein. The chemical reactions of proteins are very diverse, they are due to the presence of NH 2 -, COOH groups and radicals of various nature. These are the reactions of nitration, acylation, alkylation, esterification, oxidation-reduction and others. Proteins have acid-base, buffering, colloidal and osmotic properties.

Acid-base properties of proteins

Chemical properties. With weak heating of aqueous solutions of proteins, denaturation occurs. This will form a precipitate.

When proteins are heated with acids, hydrolysis occurs, and a mixture of amino acids is formed.

Physicochemical properties of proteins

Proteins have a high molecular weight.

The charge of a protein molecule. All proteins have at least one free —NH and —COOH group.

Protein solutions- colloidal solutions with different properties. Proteins are acidic and basic. Acidic proteins contain many glu and asp, which have extra carboxyl and fewer amino groups. Alkaline proteins contain a lot of lys and arg. Each protein molecule in an aqueous solution is surrounded by a hydration shell, since proteins have many hydrophilic groups due to amino acids (-COOH, -OH, -NH 2, -SH). In aqueous solutions, a protein molecule has a charge. The protein charge in the water can vary depending on the pH.

Protein precipitation. Proteins have a hydration shell, a charge that prevents sticking. For deposition, it is necessary to remove the hydration shell and charge.

1. Hydration. The process of hydration means the binding of water by proteins, while they exhibit hydrophilic properties: they swell, their mass and volume increase. The swelling of the protein is accompanied by its partial dissolution. The hydrophilicity of individual proteins depends on their structure. The hydrophilic amide (–CO – NH–, peptide bond), amine (NH2) and carboxyl (COOH) groups present in the composition and located on the surface of the protein macromolecule attract water molecules, strictly orienting them to the surface of the molecule. Surrounding protein globules, a hydration (water) shell prevents the stability of protein solutions. At the isoelectric point, proteins have the least ability to bind water, the hydration shell around the protein molecules is destroyed, so they combine to form large aggregates. Aggregation of protein molecules also occurs when they are dehydrated with the help of some organic solvents, for example, ethyl alcohol. This leads to the precipitation of proteins. When the pH of the medium changes, the protein macromolecule becomes charged, and its hydration capacity changes.

Precipitation reactions are divided into two types.

Salting out proteins: (NH 4) SO 4 - only the hydration shell is removed, the protein retains all types of its structure, all bonds, retains its native properties. Such proteins can then be re-dissolved and used.

Sedimentation with the loss of the native properties of the protein is an irreversible process. The hydration shell and charge are removed from the protein, various properties in the protein are disrupted. For example, salts of copper, mercury, arsenic, iron, concentrated inorganic acids - HNO 3, H 2 SO 4, HCl, organic acids, alkaloids - tannins, iodine mercury. The addition of organic solvents decreases the degree of hydration and leads to protein precipitation. Acetone is used as such solvents. Proteins are also precipitated using salts such as ammonium sulfate. The principle of this method is based on the fact that as the salt concentration in the solution increases, the ionic atmospheres formed by the protein counterions are compressed, which contributes to their convergence to a critical distance at which the intermolecular van der Waals attraction forces outweigh the Coulomb repulsive forces of the counterions. This leads to adhesion of protein particles and their precipitation.

When boiling, protein molecules begin to move chaotically, collide, charge is removed, and the hydration shell decreases.

To detect proteins in solution, the following are used:

color reactions;

precipitation reactions.

Methods for the isolation and purification of proteins.

homogenization- cells are ground to a homogeneous mass;

extraction of proteins with water or water-salt solutions;

1. salting out;

   electrophoresis;

   chromatography: adsorption, splitting;

   ultracentrifugation.

Structural organization of proteins.

Primary structure- determined by the sequence of amino acids in the peptide chain, stabilized by covalent peptide bonds (insulin, pepsin, chymotrypsin).

Secondary structure- the spatial structure of the protein. It is either -spiral or -folding. Hydrogen bonds are created.

Tertiary structure- globular and fibrillar proteins. Stabilize hydrogen bonds, electrostatic forces (СОО-, NH3 +), hydrophobic forces, sulfide bridges, are determined by the primary structure. Globular proteins - all enzymes, hemoglobin, myoglobin. Fibrillar proteins - collagen, myosin, actin.

Quaternary structure- available only in some proteins. These proteins are built from several peptides. Each peptide has its own primary, secondary, tertiary structure, called protomers. Several protomers are linked together to form one molecule. One protomer does not function as a protein, but only in conjunction with other protomers.

Example: hemoglobin = -globula + -globula - transfers O 2 in aggregate, and not separately.

Protein can renature. This requires a very short exposure to the agents.

6) Methods for detecting proteins.

Proteins are high molecular weight biological polymers, the structural (monomeric) units of which are -amino acids. The amino acids in proteins are linked to each other by a peptide bond, the formation of which occurs due to the carboxyl group standing at -carbon atom of one amino acid and -amino group of another amino acid with the release of a water molecule. Monomeric units of proteins are called amino acid residues.

Peptides, polypeptides and proteins differ not only in quantity, composition, but also in the sequence of amino acid residues, physicochemical properties and functions performed in the body. The molecular weight of proteins varies from 6 thousand to 1 million or more. The chemical and physical properties of proteins are determined by the chemical nature and physicochemical properties of the radicals, the amino acid residues included in them. Methods for detecting and quantifying proteins in biological objects and food products, as well as their isolation from tissues and biological fluids, are based on the physical and chemical properties of these compounds.

Proteins when interacting with certain chemicals give colored compounds... The formation of these compounds occurs with the participation of amino acid radicals, their specific groups or peptide bonds. Color reactions make it possible to establish the presence of protein in a biological object or solution and prove the presence certain amino acids in a protein molecule... Several methods for the quantitative determination of proteins and amino acids have been developed on the basis of color reactions.

Considered universal biuret and ninhydrin reactions, since all proteins give them. Xanthoprotein reaction, Fol's reaction and others are specific, since they are due to the radical groups of certain amino acids in the protein molecule.

Color reactions make it possible to establish the presence of protein in the test material and the presence of certain amino acids in its molecules.

Biuret reaction... The reaction is due to the presence in proteins, peptides, polypeptides peptide bonds, which in an alkaline environment form with copper (II) ions complex compounds colored in purple (with a red or blue tint) color... Coloring is due to the presence of at least two groups in the molecule -CO-NH- connected directly to each other or with the participation of a carbon or nitrogen atom.

Copper (II) ions are connected by two ionic bonds with groups = С─О ˉ and four coordination bonds with nitrogen atoms (= N―).

The intensity of the color depends on the amount of protein in the solution. This allows this reaction to be used for protein quantification. The color of the colored solutions depends on the length of the polypeptide chain. Proteins give a blue-violet color; the products of their hydrolysis (poly- and oligopeptides) are red or pink in color. The biuret reaction is produced not only by proteins, peptides and polypeptides, but also by biuret (NH 2 -CO-NH-CO-NH 2), oxamide (NH 2 -CO-CO-NH 2), histidine.

The complex compound of copper (II) with peptide groups formed in an alkaline medium has the following structure:

Ninhydrin reaction... In this reaction, solutions of protein, polypeptides, peptides and free α-amino acids, when heated with ninhydrin, give a blue, blue-violet or pink-violet color. Coloring in this reaction develops due to the α-amino group.

React very easily with ninhydrin -amino acids. Along with them, Ruemann's blue-violet is also formed by proteins, peptides, primary amines, ammonia and some other compounds. Secondary amines such as proline and hydroxyproline give a yellow color.

The ninhydrin reaction is widely used for the detection and quantification of amino acids.

Xanthoprotein reaction. This reaction indicates the presence of aromatic amino acid residues in proteins - tyrosine, phenylalanine, tryptophan. Based on the nitration of the benzene ring of the radicals of these amino acids with the formation of nitro compounds, colored yellow (Greek "Xanthos" - yellow). Using tyrosine as an example, this reaction can be described in the form of the following equations.

In an alkaline environment, nitro-derivatives of amino acids form salts of a quinoid structure, colored orange. The xantoprotein reaction is given by benzene and its homologues, phenol and other aromatic compounds.

Reactions to amino acids containing a thiol group in a reduced or oxidized state (cysteine, cystine).

Fol's reaction. When boiling with alkali, sulfur is easily split off from cysteine ​​in the form of hydrogen sulfide, which forms sodium sulfide in an alkaline medium:

In this regard, the reactions for the determination of thiol-containing amino acids in solution are divided into two stages:

Sulfur transition from organic to inorganic state

Detection of sulfur in solution

To detect sodium sulfide, lead acetate is used, which, when interacting with sodium hydroxide, turns into its plumbite:

Pb (CH 3 COO) 2 + 2NaOH Pb (ONa) 2 + 2CH 3 COOH

As a result of the interaction of sulfur and lead ions, lead sulfide of black or brown color is formed:

Na 2 S + Pb(ONa) 2 + 2 H 2 O PbS (black sediment) + 4NaOH

To determine sulfur-containing amino acids, an equal volume of sodium hydroxide and a few drops of a solution of lead acetate are added to the test solution. With intensive boiling for 3-5 minutes, the liquid turns black.

The presence of cystine can be determined by this reaction, since cystine is readily reduced to cysteine.

Millon's reaction:

This is a reaction to the amino acid tyrosine.

Free phenolic hydroxyls of tyrosine molecules, when interacting with salts, give compounds of the mercury salt of the nitro derivative of tyrosine, colored pinkish-red:

Pauli's reaction to histidine and tyrosine . Pauli's reaction makes it possible to detect in the protein the amino acids histidine and tyrosine, which form cherry-red complex compounds with diazobenzenesulfonic acid. Diazobenzenesulfonic acid is formed in the diazotization reaction when sulfanilic acid interacts with sodium nitrite in an acidic medium:

An equal volume of an acidic solution of sulfanilic acid (prepared using hydrochloric acid) and a double volume of a sodium nitrite solution are added to the test solution, mix thoroughly and immediately add soda (sodium carbonate). After stirring, the mixture turns cherry red, provided that histidine or tyrosine is present in the test solution.

Reaction of Adamkevich-Hopkins-Kohl (Schultz-Raspail) to tryptophan (reaction to the indole group). Tryptophan reacts in an acidic environment with aldehydes, forming colored condensation products. The reaction takes place due to the interaction of the indole ring of tryptophan with an aldehyde. It is known that formaldehyde is formed from glyoxylic acid in the presence of sulfuric acid:

R
Solutions containing tryptophan give a red-violet coloration in the presence of glyoxylic and sulfuric acids.

Glyoxylic acid is always present in small amounts in glacial acetic acid. Therefore, the reaction can be carried out using acetic acid. At the same time, an equal volume of glacial (concentrated) acetic acid is added to the test solution and carefully heated until the precipitate dissolves. After cooling, a volume of concentrated sulfuric acid equal to the added volume of glyoxylic acid is added to the mixture carefully along the wall (to avoid mixing liquids). After 5-10 minutes, the formation of a red-violet ring is observed at the interface between the two layers. If you mix the layers, the contents of the dish will turn evenly purple.

TO

ondensation of tryptophan with formaldehyde:

The condensation product is oxidized to bis-2-tryptophanylcarbinol, which in the presence of mineral acids forms blue-violet salts:

7) Classification of proteins. Methods for studying the amino acid composition.

A strict nomenclature and classification of proteins still does not exist. The names of proteins are given on random grounds, most often taking into account the source of protein isolation or taking into account its solubility in certain solvents, the shape of the molecule, etc.

The classification of proteins is carried out according to composition, particle shape, solubility, amino acid composition, origin, etc.

1. By composition proteins are divided into two large groups: simple and complex proteins.

Simple (proteins) include proteins that give only amino acids during hydrolysis (proteinoids, protamines, histones, prolamins, glutelins, globulins, albumin). Silk fibroin, egg serum albumin, pepsin, etc. are examples of simple proteins.

Complex proteins (proteins) include proteins composed of a simple protein and an additional (prosthetic) group of non-protein nature. The group of complex proteins is divided into several subgroups depending on the nature of the non-protein component:

Metalloproteins containing metals (Fe, Cu, Mg, etc.) associated directly with the polypeptide chain;

Phosphoproteins - contain residues of phosphoric acid, which are attached by ester bonds to the protein molecule at the site of the hydroxyl groups of serine, threonine;

Glycoproteins - their prosthetic groups are carbohydrates;

Chromoproteins - consist of a simple protein and a colored non-protein compound associated with it, all chromoproteins are biologically very active; as prosthetic groups they can be derivatives of porphyrin, isoalloxazine and carotene;

Lipoproteins - a prosthetic group of lipids - triglycerides (fats) and phosphatides;

Nucleoproteins are proteins consisting of a simple protein and a nucleic acid attached to it. These proteins play a colossal role in the life of the body and will be discussed below. They are part of any cell, some nucleoproteins exist in nature in the form of special particles with pathogenic activity (viruses).

2. By particle shape- proteins are divided into fibrillar (filamentous) and globular (spherical) (see page 30).

3. By solubility and characteristics of the amino acid composition the following groups of simple proteins are distinguished:

Proteinoids are proteins of supporting tissues (bones, cartilage, ligaments, tendons, hair, nails, skin, etc.). These are mainly fibrillar proteins with a high molecular weight (> 150,000 Da), insoluble in common solvents: water, salt and water-alcohol mixtures. They dissolve only in specific solvents;

Protamines (the simplest proteins) are proteins that are soluble in water and contain 80-90% arginine and a limited set (6-8) of other amino acids, presented in the milk of various fish. Due to the high content of arginine, they have basic properties, their molecular weight is relatively low and approximately equal to 4000-12000 Da. They are a protein component in nucleoproteins;

Histones are highly soluble in water and dilute solutions of acids (0.1 N), are distinguished by a high content of amino acids: arginine, lysine and histidine (at least 30%) and therefore have basic properties. These proteins are found in significant quantities in the nuclei of cells as part of nucleoproteins and play an important role in the regulation of nucleic acid metabolism. The molecular weight of histones is small and equal to 11000-24000 Da;

Globulins are proteins that are insoluble in water and salt solutions with a salt concentration of more than 7%. Globulins completely precipitate at 50% saturation of the solution with ammonium sulfate. These proteins have a high glycine content (3.5%), their molecular weight> 100,000 Da. Globulins - slightly acidic or neutral proteins (p1 = 6-7.3);

Albumins are proteins that are readily soluble in water and strong salt solutions, and the concentration of salt (NH 4) 2 S0 4 should not exceed 50% of saturation. At higher concentrations, the albumin is salted out. Compared to globulins, these proteins contain three times less glycine and have a molecular weight of 40,000-70000 Da. Albumin has an excessive negative charge and acidic properties (pl = 4.7) due to the high content of glutamic acid;

Prolamins are a group of plant proteins found in the gluten of cereal plants. They are soluble only in 60-80% aqueous solution of ethyl alcohol. Prolamins have a characteristic amino acid composition: they contain a lot (20-50%) of glutamic acid and proline (10-15%), which is why they got their name. Their molecular weight is over 100,000 Da;

Glutelins are vegetable proteins insoluble in water, salt solutions and ethanol, but soluble in dilute (0.1 N) solutions of alkalis and acids. In terms of amino acid composition and molecular weight, they are similar to prolamins, but they contain more arginine and less proline.

Methods for studying the amino acid composition

Under the action of enzymes of digestive juices, proteins are broken down into amino acids. Two important conclusions were made: 1) the composition of proteins includes amino acids; 2) by the methods of hydrolysis, the chemical, in particular, amino acid, composition of proteins can be studied.

To study the amino acid composition of proteins, a combination of acidic (HCl), alkaline [Ba (OH) 2] and, less often, enzymatic hydrolysis, or one of them, is used. It was found that during the hydrolysis of a pure protein that does not contain impurities, 20 different α-amino acids are released. All other amino acids discovered in the tissues of animals, plants and microorganisms (more than 300) exist in nature in a free state or in the form of short peptides or complexes with other organic substances.

The first step in determining the primary structure of proteins is to qualitatively and quantitatively assess the amino acid composition of a given individual protein. It must be remembered that for research you need to have a certain amount of pure protein, without admixtures of other proteins or peptides.

Acid hydrolysis of protein

To determine the amino acid composition, it is necessary to destroy all peptide bonds in the protein. The analyzed protein is hydrolyzed in 6 mol / L HC1 at a temperature of about 110 ° C for 24 hours. As a result of this treatment, peptide bonds in the protein are destroyed, and only free amino acids are present in the hydrolyzate. In addition, glutamine and asparagine are hydrolyzed to glutamic and aspartic acids (i.e., the amide bond in the radical is broken and the amino group is split off from them).

Separation of amino acids using ion exchange chromatography

The mixture of amino acids obtained by acid hydrolysis of proteins is separated in a column with a cation exchange resin. Such a synthetic resin contains negatively charged groups firmly bound to it (for example, sulfonic acid residues -SO 3 -), to which Na + ions are attached (Fig. 1-4).

A mixture of amino acids is introduced into the cation exchanger in an acidic medium (pH 3.0), where the amino acids are mainly cations, i.e. carry a positive charge. The positively charged amino acids bind to the negatively charged resin particles. The higher the total charge of an amino acid, the stronger its bond with the resin. Thus, the amino acids lysine, arginine and histidine bind most strongly to the cation exchanger, while aspartic and glutamic acids are the weakest.

The release of amino acids from the column is carried out by elution (elution) with a buffer solution with increasing ionic strength (i.e., with increasing NaCl concentration) and pH. With an increase in pH, amino acids lose a proton, as a result, their positive charge decreases, and hence the bond strength with negatively charged resin particles.

Each amino acid leaves the column at a specific pH and ionic strength. Collecting the solution (eluate) from the lower end of the column in small portions, it is possible to obtain fractions containing individual amino acids.

(for more details "hydrolysis" see question No. 10)

8) Chemical bonds in the structure of the protein.

9) The concept of the hierarchy and structural organization of proteins. (see question number 12)

10) Protein hydrolysis. The chemistry of the reaction (stepwise, catalysts, reagents, reaction conditions) is a complete description of hydrolysis.

11) Chemical transformations of proteins.

Denaturation and renaturation

When protein solutions are heated to 60-80% or under the action of reagents that destroy non-covalent bonds in proteins, the tertiary (quaternary) and secondary structure of the protein molecule is destroyed, it takes, to a greater or lesser extent, the form of a random random coil. This process is called denaturation. Acids, alkalis, alcohols, phenols, urea, guanidine chloride, etc. can be used as denaturing reagents. The essence of their action is that they form hydrogen bonds with = NH and = CO - groups of the peptide backbone and with acid groups of amino acid radicals, replacing their own intramolecular hydrogen bonds in the protein as a result of which the secondary and tertiary structures change. During denaturation, the solubility of the protein decreases, it "coagulates" (for example, when boiling a chicken egg), the biological activity of the protein is lost. This is the basis, for example, of the use of an aqueous solution of carbolic acid (phenol) as an antiseptic. Under certain conditions, with slow cooling of the denatured protein solution, renaturation occurs - restoration of the original (native) conformation. This confirms the fact that the nature of the folding of the peptide chain is determined by the primary structure.

The process of denaturation of an individual protein molecule, leading to the disintegration of its "rigid" three-dimensional structure, is sometimes called the melting of the molecule. Almost any noticeable change in external conditions, for example, heating or a significant change in pH, leads to a sequential disruption of the quaternary, tertiary and secondary structures of the protein. Usually denaturation is caused by an increase in temperature, the action of strong acids and alkalis, salts of heavy metals, some solvents (alcohol), radiation, etc.

Denaturation often leads to the process of aggregation of protein particles into larger ones in the colloidal solution of protein molecules. Visually, it looks like, for example, the formation of "protein" when frying eggs.

Renaturation is the reverse process of denaturation, in which proteins return to their natural structure. It should be noted that not all proteins are capable of renaturing; for most proteins, denaturation is irreversible. If, during protein denaturation, physicochemical changes are associated with the transition of the polypeptide chain from a densely packed (ordered) state to a disordered state, then during renaturation, the ability of proteins to self-organization is manifested, the path of which is predetermined by the sequence of amino acids in the polypeptide chain, that is, its primary structure, determined by hereditary information ... In living cells, this information is probably crucial for the transformation of a disordered polypeptide chain during or after its biosynthesis on the ribosome into the structure of a native protein molecule. When double-stranded DNA molecules are heated to a temperature of about 100 ° C, the hydrogen bonds between the bases are broken, and the complementary strands diverge - the DNA denatures. However, upon slow cooling, the complementary strands can rejoin to form a regular double helix. This renaturation ability of DNA is used to produce artificial hybrid DNA molecules.

Natural protein bodies are endowed with a specific, strictly specified spatial configuration and have a number of characteristic physicochemical and biological properties at physiological values ​​of temperature and pH of the environment. Under the influence of various physical and chemical factors, proteins are coagulated and precipitated, losing their native properties. Thus, denaturation should be understood as a violation of the general plan of the unique structure of the native protein molecule, mainly its tertiary structure, leading to the loss of its characteristic properties (solubility, electrophoretic mobility, biological activity, etc.). Most proteins denature when their solutions are heated 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 number of free functional SH-groups, and a change in the nature of X-ray scattering. The most characteristic sign of denaturation is a sharp decrease or complete loss of the protein's biological activity (catalytic, antigenic or hormonal). Protein denaturation caused by 8M urea or another agent destroys mainly non-covalent bonds (in particular, hydrophobic interactions and hydrogen bonds). Disulfide bonds in the presence of the reducing agent mercaptoethanol are broken, while the peptide bonds of the polypeptide chain itself are not affected. Under these conditions, globules of native protein molecules unfold and random and disordered structures are formed (Fig.)

Denaturation of a protein molecule (scheme).

a - initial state; b - incipient reversible violation of the molecular structure; c - irreversible unfolding of the polypeptide chain.

Denaturation and renaturation of ribonuclease (according to Anfinsen).

a - deployment (urea + mercaptoethanol); b - re-folding.

1. Protein hydrolysis: H +

[- NH2─CH─ CO─NH─CH─CO -] n + 2nH2O → n NH2 - CH - COOH + n NH2 ─ CH ─ COOH

│ │ ‌‌│ │

Amino acid 1 amino acid 2

2. Protein precipitation:

a) reversible

Protein in solution ↔ protein precipitate. It occurs under the action of solutions of salts Na +, K +

b) irreversible (denaturation)

During denaturation under the influence of external factors (temperature; mechanical action - pressure, rubbing, shaking, ultrasound; the action of chemical agents - acids, alkalis, etc.), the secondary, tertiary and quaternary structures of the protein macromolecule change, that is, its native spatial structure. The primary structure, and therefore the chemical composition of the protein, does not change.

Denaturation changes the physical properties of proteins: solubility decreases, biological activity is lost. At the same time, the activity of some chemical groups increases, the effect on proteins of proteolytic enzymes is facilitated, and, therefore, it is easier to hydrolyze.

For example, albumin - egg white - precipitates from solution (coagulates) at a temperature of 60-70 °, losing its ability to dissolve in water.

Diagram of the process of protein denaturation (destruction of the tertiary and secondary structures of protein molecules)

3. Combustion of proteins

Proteins burn with the formation of nitrogen, carbon dioxide, water, and some other substances. Burning is accompanied by a characteristic smell of burnt feathers

4. Colored (qualitative) reactions to proteins:

a) xanthoprotein reaction (to amino acid residues containing benzene rings):

Protein + HNO3 (conc.) → yellow color

b) biuret reaction (to peptide bonds):

Protein + CuSO4 (sat) + NaOH (conc) → bright purple coloration

c) cysteine ​​reaction (for amino acid residues containing sulfur):

Protein + NaOH + Pb (CH3COO) 2 → Black staining

Proteins are the basis of all life on Earth and perform diverse functions in organisms.

Salting out proteins

Salting out is the process of isolating proteins from aqueous solutions with neutral solutions of concentrated salts of alkali and alkaline earth metals. When high concentrations of salts are added to the protein solution, the protein particles are dehydrated and the charge is removed, while the proteins precipitate. The degree of protein precipitation depends on the ionic strength of the precipitant solution, the particle size of the protein molecule, the magnitude of its charge, and hydrophilicity. Different proteins are precipitated at different salt concentrations. Therefore, in sediments obtained by a gradual increase in the concentration of salts, individual proteins are found in different fractions. The salting-out of proteins is a reversible process, and after the removal of the salt, the protein regains its natural properties. Therefore, salting out is used in clinical practice for the separation of serum proteins, as well as for the isolation and purification of various proteins.

The added anions and cations destroy the hydrated protein shell of proteins, which is one of the factors of stability of protein solutions. The most commonly used solutions of Na and ammonium sulfates. 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 (when a 50% solution of ammonium sulfate (NH4) 2SO4 is added, a precipitate is formed) and albumin (when a 100% solution of ammonium sulfate (NH4) 2SO4 is added, a precipitate is formed).

The salting out value is influenced by:

1) the nature and concentration of salt;

2) pH-environment;

3) temperature.

In this case, the valences of the ions play the main role.

12) Features of the organization of the primary, secondary, tertiary structure of the protein.

At present, the existence of four levels of the structural organization of a protein molecule has been experimentally proven: primary, secondary, tertiary and quaternary structure.