Among the many millions of types of molecules that make up the biochemical environment of the body, there are many thousands that play an informational role. Even if we do not consider those substances that the body releases into the environment, communicating itself to other living beings: fellow tribesmen, enemies and victims, a huge variety of molecules can be classified into different classes biologically active substances(abbreviated as BAS), circulating in the liquid media of the body and transmitting this or that information from the center to the periphery, from one cell to another, or from the periphery to the center. Despite the diversity of composition and chemical structure, all these molecules in one way or another directly affect the metabolic processes carried out by specific cells of the body.
The most important for the physiological regulation of biologically active substances are mediators, hormones, enzymes and vitamins.
Mediators - These are substances of non-protein nature, having a relatively simple structure and low molecular weight. They are released by the endings of nerve cells under the influence of the next nerve impulse received there (from special vesicles in which they accumulate in the intervals between nerve impulses). Depolarization of the nerve fiber membrane leads to the rupture of the mature vesicle, and drops of the transmitter enter the synaptic cleft. A synapse is the junction of two nerve fibers or a nerve fiber with a cell of another tissue. Although the signal is transmitted electrically along a nerve fiber, unlike conventional metal wires, nerve fibers cannot simply be mechanically connected to each other: an impulse cannot be transmitted in this way, since the nerve fiber sheath is not a conductor, but an insulator. In this sense, the nerve fiber is less like a wire and more like a cable surrounded by a layer of electrical insulator. This is why a chemical mediator is needed. This role is precisely performed by the mediator molecule. Once in the synaptic cleft, the transmitter acts on the postsynaptic membrane, leading to a local change in its polarization, and thus an electrical impulse is generated in the cell to which the excitation needs to be transferred. Most often, the molecules of acetylcholine, adrenaline, norepinephrine, dopamine and gamma-aminobutyric acid (GABA) act as mediators in the human body. As soon as the action of the mediator on the postsynaptic membrane is completed, the mediator molecule is destroyed with the help of special enzymes that are constantly present at this cell junction, thus preventing overexcitation of the postsynaptic membrane and, accordingly, the cells on which the informational influence is exerted. It is for this reason that one impulse reaching the presynaptic membrane generates a single impulse in the postsynaptic membrane. Depletion of transmitter reserves in the presynaptic membrane can sometimes cause disturbances in the conduction of nerve impulses.
Hormones - high molecular weight substances produced by the endocrine glands to control the activity of other organs and systems of the body.
According to their chemical composition, hormones can belong to different classes. organic compounds, significantly different in molecular size (Table 13). Chemical composition The hormone determines the mechanism of its interaction with target cells.
Hormones can be of two types - direct acting or tropic. The former directly affect somatic cells, changing their metabolic state and causing them to change their functional activity. The latter are intended to influence other endocrine glands, in which, under the influence of tropic hormones, the production of their own hormones, which usually act directly on somatic cells, is accelerated or slowed down.
Federal Agency for Education
State educational institution
higher professional education "Perm State Technical University" Department of Chemistry and Biotechnology
Chemistry of biologically active compounds
Lecture notes for full-time students
specialty 070100 “Biotechnology”
Publishing house
Perm State Technical University
Compiled by: Ph.D. Biol. Science L.V. Anikina
Reviewer
Ph.D. chem. Sciences, Associate Professor I.A. Tolmacheva
(Perm State University)
Chemistry of biologically active substances/comp. L.V. Anikina - Perm: Perm Publishing House. state tech. University, 2009. – 109 p.
Lecture notes on the course program “Chemistry of biologically active substances” are presented.
Intended for full-time students in the direction 550800 “Chemical technology and biotechnology”, specialty 070100 “Biotechnology”.
© State Educational Institution of Higher Professional Education
"Perm State
Technical University", 2009
Introduction…………………………………………………………………………………..4
Lecture 1. Chemical components of living things…………………………………….7
Lecture 2. Carbohydrates…………………………………………………………….12
Lecture 3. Lipids………………………………………………………………..20
Lecture 4. Amino acids……………………………………………………..…35
Lecture 5. Proteins……………………………………………………………….….43
Lecture 6. Properties of proteins……………………………………………………...57
Lecture 7. Simple and complex proteins……………………………………………………...61
Lecture 8. Nucleic acids and nucleoproteins………………………….72
Lecture 9. Enzymes……………………………………………………….….85
Lecture 10. Classification of enzymes………………………………………………………... 94
Introduction
When training specialists in biotechnology, the most important basic disciplines are biochemistry, organic chemistry and the chemistry of biologically active substances. These disciplines form the fundamental basis of biotechnology, the development of which is associated with the solution of such major social problems of our time as the provision of energy, feed and food resources, environmental protection and human health.
According to the requirements of the State Standard of Higher Professional Education for the mandatory minimum content of basic educational programs in the direction 550800 “Chemical technology and biotechnology”, specialty 070100 “Biotechnology”, the discipline “Chemistry of biologically active substances” includes the following didactic units: structure and spatial organization of proteins, nucleic acids acids, carbohydrates, lipids, low molecular weight bioregulators and antibiotics; concept of enzymes, antibodies, structural proteins; enzymatic catalysis.
The purpose of teaching the discipline “Chemistry of Biologically Active Substances” is to form students’ ideas about the structure and fundamentals of functioning of biologically active substances, about enzymatic catalysis.
Lectures on the discipline “Chemistry of Biologically Active Substances” are based on students’ knowledge of the courses “General Chemistry”, “Inorganic Chemistry”, “Physical Chemistry”, “Analytical Chemistry” and “Chemistry of Coordination Compounds”. The provisions of this discipline are used for further study of the courses “Biochemistry”, “Microbiology”, “Biotechnology”.
The proposed lecture notes cover the following topics taught in the course “Chemistry of Biologically Active Substances”:
Carbohydrates, classification, chemical structure and biological role, chemical reactions characteristic of carbohydrates. Monosaccharides, disaccharides, polysaccharides.
Lipids. Classification by chemical structure, biological functions of lipids and their derivatives - vitamins, hormones, bioregulators.
Amino acids, general formula, classification and biological role. Physicochemical properties of amino acids. Proteinogenic amino acids, amino acids as precursors of biologically active molecules - coenzymes, bile acids, neurotransmitters, hormones, histohormones, alkaloids, and some antibiotics.
Proteins, elemental composition and functions of proteins. Primary structure of a protein. Characteristics of the peptide bond. Protein secondary structure: α-helix and β-sheet.
Supersecondary protein structure, domain principle of protein evolution. Tertiary structure of a protein and the bonds that stabilize it.
The concept of fibrillar and globular proteins. Quaternary structure of protein.
Physicochemical and biological properties of proteins.
Nucleic acids, biological role in the cell.
Nitrogen bases, nucleosides, nucleotides, polynucleotides of DNA and RNA.
Types of RNA. Spatial structure of DNA, levels of DNA compaction in chromatin.
Enzymes as biological catalysts, their difference from non-protein catalysts.
Simple and complex enzymes. The active site of the enzyme. The mechanism of action of enzymes, reduction of activation energy, formation of an enzyme-substrate complex, theory of bond deformation, acid-base and covalent catalysis.
Enzyme isoforms. Multienzyme systems. Regulation of enzyme activity at the cellular level: limited proteolysis, molecular aggregation, chemical modification, allosteric inhibition. Types of inhibition: reversible and irreversible, competitive and non-competitive. Enzyme activators and inhibitors. Nomenclature of enzymes. International classification of enzymes.
Oxidoreductases: NAD-dependent dehydrogenases, flavin-dependent dehydrogenases, quinones, cytochrome system, oxidases.
Transferases: phosphotransferases, acyltransferases and coenzyme A, aminotransferases using pyridoxal phosphate, C 1 -transferases containing active forms as coenzymes
folic acid
and cyanocobalamin, a glycosyltransferase.
Hydrolases: esterases, phosphatases, glycosidases, peptidases, amidases.
Lyases: decarboxylases using thiamine pyrophosphate as a coenzyme, aldolase, hydratases, deaminases, synthases. .
Isomerases: transfer of hydrogen, phosphate and acyl groups, movement of double bonds, stereoisomerases. :
Ligases: the relationship between synthesis and the breakdown of ATP, carboxylase and the role of carboxybiotin, acyl-coenzyme A synthetase.
At the end of the lecture notes there is a list of literature that must be used to successfully master the course “Chemistry of Biologically Active Substances”.
Nonspecific metabolites Specific metabolites
A). tissue hormones (parahormones); b). true hormones.
Nonspecific metabolites - metabolic products produced by any cell in the process of vital activity and possessing biological activity (CO 2, lactic acid). Specific metabolites
- waste products produced by certain specialized types of cells, possessing biological activity and specificity of action: A) tissue hormones
Participation of biologically active substances at various levels of neurohumoral regulation:
I level : local or local regulation Provided by humoral factors : mostly - nonspecific metabolites and to a lesser extent - specific metabolites (tissue hormones).
II level of regulation : regional (organ).tissue hormones.
III level - interorgan, intersystem regulation. Humoral regulation is represented endocrine glands.
Level IV. Whole organism level. Nervous and humoral regulation are subordinated at this level of behavioral regulation.
Regulatory influence at any level is determined by a number of factors:
quantity biologically active substance;
2. quantity receptors;
3. sensitivity receptors.
In its turnsensitivity depends:
A). from functional state cells;
b). on the state of the microenvironment (pH, ion concentration, etc.);
V). on the duration of exposure to the disturbing factor.
Local regulation (1 level of regulation)
Wednesday is tissue fluid. Main factors:
Creative connections.
2. Nonspecific metabolites.
Creative connections- exchange between cells of macromolecules that carry information about cellular processes, allowing tissue cells to function cooperatively. This is one of the most evolutionarily old methods of regulation.
Keylons- substances that provide creative connections. They are represented by simple proteins or glycoproteins that influence cell division and DNA synthesis. Violation of creative connections may underlie a number of diseases (tumor growth) as well as the aging process.
Nonspecific metabolites - CO 2, lactic acid - act at the site of formation on neighboring groups of cells.
Regional (organ) regulation (2nd level of regulation)
1. nonspecific metabolites,
2. specific metabolites (tissue hormones).
Tissue hormone system
|
Substance |
Place of generation |
Effect |
|
Seratonin |
intestinal mucosa (enterochromaffin tissue), brain, platelets |
CNS mediator, vasoconstrictor effect, vascular-platelet hemostasis |
|
Prostaglandins |
derivative of arachidonic and linolenic acid, body tissue |
The vasomotor effect, and the dilator and constrictor effect, enhances uterine contractions, enhances the excretion of water and sodium, reduces the secretion of enzymes and HCl by the stomach |
|
Bradykinin |
Peptide, blood plasma, salivary glands, lungs |
vasodilator effect, increases vascular permeability |
|
Acetylcholine |
brain, ganglia, neuromuscular junctions |
relaxes the smooth muscles of blood vessels, reduces heart contractions |
|
Histamine |
histidine derivative, stomach and intestines, skin, mast cells, basophils |
mediator of pain receptors, dilates microvessels, increases the secretion of gastric glands |
|
Endorphins, enkephalins |
brain |
analgesic and adaptive effects |
|
Gastrointestinal hormones |
are produced in various departments Gastrointestinal tract |
participate in the regulation of secretion, motility and absorption processes |
Doctor of Biological Sciences, Professor V. M. Shkumatov;
deputy general director on questions
innovative development of RUE "Belmedpreparaty"
Candidate of Technical Sciences T. V. Trukhacheva
Leontyev, V. N.
Chemistry of biologically active substances: an electronic course of lecture texts for students of the specialty 1-48 02 01 “Biotechnology” of full-time and part-time forms of study / V. N. Leontiev, O. S. Ignatovets. – Minsk: BSTU, 2013. – 129 p.
The electronic course of lecture texts is devoted to the structural and functional features and chemical properties of the main classes of biologically active substances (proteins, carbohydrates, lipids, vitamins, antibiotics, etc.). Methods of chemical synthesis and structural analysis of the listed classes of compounds, their properties and effects on biological systems, as well as distribution in nature.
| Topic 1. Introduction | 4 |
| Topic 2. Proteins and peptides. Primary structure of proteins and peptides | |
| Topic 3. Structural organization of proteins and peptides. Selection methods | |
| Topic 4. Chemical synthesis and chemical modification of proteins and peptides | |
| Topic 5. Enzymes | 45 |
| Topic 6. Some biologically important proteins | 68 |
| Topic 7. Structure of nucleic acids | 76 |
| Topic 8. Structure of carbohydrates and carbohydrate-containing biopolymers | |
| Topic 9. Structure, properties and chemical synthesis of lipids | 104 |
| Topic 10. Steroids | 117 |
| Topic 11. Vitamins | 120 |
| Topic 12. Introduction to pharmacology. Pharmacokinetics | 134 |
| Topic 13. Antimalarial drugs | 137 |
| Topic 14. Means affecting the central nervous system | |
| Topic 15. Sulfonamide drugs | 144 |
| Topic 16. Antibiotics | 146 |
| Bibliography | 157 |
Topic 1. Introduction
The chemistry of biologically active substances studies the structure and biological functions of the most important components of living matter, primarily biopolymers and low-molecular bioregulators, focusing on elucidating the patterns of the relationship between structure and biological action. Essentially, it is the chemical foundation modern biology. By developing the fundamental problems of the chemistry of the living world, bioorganic chemistry contributes to solving the problems of obtaining practically important drugs for medicine, agriculture, and a number of industries.
Objects of study: proteins and peptides, nucleic acids, carbohydrates, lipids, mixed biopolymers - glycoproteins, nucleoproteins, lipoproteins, glycolipids, etc.; alkaloids, terpenoids, vitamins, antibiotics, hormones, prostaglandins, growth substances, pheromones, toxins, as well as synthetic medications, pesticides, etc.
Research methods: the main arsenal consists of methods organic chemistry, however, to solve structural and functional problems, various physical, physicochemical, mathematical and biological methods.
Main goals: isolation of the studied compounds in an individual state using crystallization, distillation, various types of chromatography, electrophoresis, ultrafiltration, ultracentrifugation, countercurrent distribution, etc.; establishment of structure, including spatial structure, based on approaches of organic and physical-organic chemistry using mass spectrometry, various types of optical spectroscopy (IR, UV, laser, etc.), X-ray diffraction analysis, nuclear magnetic resonance, electron paramagnetic resonance, optical dispersion rotation and circular dichroism, fast kinetics methods, etc. in combination with computer calculations; chemical synthesis and chemical modification of the studied compounds, including complete synthesis, synthesis of analogues and derivatives, in order to confirm the structure, clarify the relationship between structure and biological function, and obtain practically valuable drugs; biological testing of the resulting compounds in vitro And in vivo.
Most common in biomolecules functional groups:
| hydroxyl (alcohols) |  amino group (amines) |
 aldehydic (aldehydes) |  amide (amides) |
 carbonyl (ketones) |  ester |
 carboxylic (acid) |  ethereal |
 sulfhydryl (thiols) |  methyl |
 disulfide |  ethyl |
 phosphate |  phenyl |
 guanidine |  imidazole |
Topic 2. Proteins and peptides. Primary structure of proteins and peptides
Squirrels– high molecular weight biopolymers built from amino acid residues. The molecular weight of proteins ranges from 6,000 to 2,000,000 Da. It is proteins that are the product of genetic information transmitted from generation to generation and carry out all life processes in the cell. These amazingly diverse polymers have some of the most important and versatile cellular functions.
Proteins can be divided:
1) by structure
:
simple proteins are built from amino acid residues and, upon hydrolysis, decompose only into free amino acids or their derivatives.
Complex proteins are two-component proteins that consist of a simple protein and a non-protein component called a prosthetic group. During the hydrolysis of complex proteins, in addition to free amino acids, a non-protein part or its breakdown products are formed. They may contain metal ions (metalloproteins), pigment molecules (chromoproteins), they can form complexes with other molecules (lipo-, nucleo-, glycoproteins), and also covalently bind inorganic phosphate (phosphoproteins);
2. water solubility:
– water soluble,
– salt-soluble,
– alcohol-soluble,
– insoluble;
3. functions performed : The biological functions of proteins include:
– catalytic (enzymatic),
– regulatory (ability to regulate speed chemical reactions in the cell and the level of metabolism in the whole organism),
– transport (transport of substances in the body and their transfer through biomembranes),
– structural (composed of chromosomes, cytoskeleton, connective, muscle, supporting tissues),
– receptor (interaction of receptor molecules with extracellular components and initiation of a specific cellular response).
In addition, proteins perform protective, storage, toxic, contractile and other functions;
4) depending on the spatial structure:
– fibrillar (they are used by nature as a structural material),
– globular (enzymes, antibodies, some hormones, etc.).
AMINO ACIDS, THEIR PROPERTIES
Amino acids are called carboxylic acids containing an amino group and a carboxyl group. Natural amino acids are 2-aminocarboxylic acids, or α-amino acids, although there are amino acids such as β-alanine, taurine, γ-aminobutyric acid. IN general case The α-amino acid formula looks like this: 
α-amino acids have four different substituents at the 2nd carbon atom, i.e. all α-amino acids, except glycine, have an asymmetric (chiral) carbon atom and exist in the form of two enantiomers - L- And D-amino acids. Natural amino acids are L-row. D-amino acids are found in bacteria and peptide antibiotics.
All amino acids in aqueous solutions can exist in the form of bipolar ions, and their total charge depends on the pH of the medium. The pH value at which the total charge is zero is called isoelectric point. At the isoelectric point, the amino acid is a zwitterion, i.e. its amine group is protonated, and its carboxyl group is dissociated. In the neutral pH region, most amino acids are zwitterions: 
Amino acids do not absorb light in the visible region of the spectrum, aromatic amino acids absorb light in the UV region of the spectrum: tryptophan and tyrosine at 280 nm, phenylalanine at 260 nm.
Proteins give a number of color reactions due to the presence of certain amino acid residues or general chemical groups. These reactions are widely used for analytical purposes. Among them, the most famous are the ninhydrin reaction, which allows for the quantitative determination of amino groups in proteins, peptides and amino acids, as well as the biuret reaction, used for the qualitative and quantitative determination of proteins and peptides. When a protein or peptide, but not an amino acid, is heated with CuSO 4 in an alkaline solution, a violet-colored copper complex compound is formed, the amount of which can be determined spectrophotometrically. Color reactions to individual amino acids are used to detect peptides containing the corresponding amino acid residues. To identify the guanidine group of arginine, the Sakaguchi reaction is used - when interacting with a-naphthol and sodium hypochlorite, guanidines in alkaline environment give a red color. The indole ring of tryptophan can be detected by the Ehrlich reaction - a red-violet color when reacted with p-dimethylamino-benzaldehyde in H 2 SO 4. The Pauli reaction reveals histidine and tyrosine residues, which in alkaline solutions react with diazobenzene sulfonic acid, forming red-colored derivatives.
Biological role of amino acids:
1) structural elements of peptides and proteins, so-called proteinogenic amino acids. Proteins contain 20 amino acids, which are encoded by the genetic code and incorporated into proteins during translation, some of which can be phosphorylated, acylated or hydroxylated;
2) structural elements of other natural compounds - coenzymes, bile acids, antibiotics;
3) signaling molecules. Some of the amino acids are neurotransmitters or precursors of neurotransmitters, hormones and histohormones;
4) the most important metabolites, for example, some amino acids are precursors of plant alkaloids, or serve as nitrogen donors, or are vital components of nutrition.
The nomenclature, molecular weight and pK values of amino acids are given in Table 1.
Table 1
Nomenclature, molecular weight and pK values of amino acids
| Amino acid | Designation | Molecular weight | p K 1 (−COOH) | p K 2 (−NH3+) | p K R (R-groups) |
| Glycine | Gly G | 75 | 2,34 | 9,60 | − |
| Alanin | Ala A | 89 | 2,34 | 9,69 | − |
| Valin | Val V | 117 | 2,32 | 9,62 | − |
| Leucine | Leu L | 131 | 2,36 | 9,60 | − |
| Isoleucine | Ile I | 131 | 2,36 | 9,68 | − |
| Proline | Pro P | 115 | 1,99 | 10,96 | − |
| Phenylalanine | PheF | 165 | 1,83 | 9,13 | − |
| Tyrosine | Tyr Y | 181 | 2,20 | 9,11 | 10,07 |
| Tryptophan | Trp W | 204 | 2,38 | 9,39 | − |
| Serin | Ser S | 105 | 2,21 | 9,15 | 13,60 |
| Threonine | Thr T | 119 | 2,11 | 9,62 | 13,60 |
| Cysteine | Cys C | 121 | 1,96 | 10,78 | 10,28 |
| Methionine | Met M | 149 | 2,28 | 9,21 | − |
| Asparagine | Asn N | 132 | 2,02 | 8,80 | − |
| Glutamine | Gln Q | 146 | 2,17 | 9,13 | − |
| Aspartate | Asp D | 133 | 1,88 | 9,60 | 3,65 |
| Glutamate | Glu E | 147 | 2,19 | 9,67 | 4,25 |
| Lysine | Lys K | 146 | 2,18 | 8,95 | 10,53 |
| Arginine | Arg R | 174 | 2,17 | 9,04 | 12,48 |
| Histidine | His H | 155 | 1,82 | 9,17 | 6,00 |
Amino acids vary in solubility in water. This is due to their zwitterionic nature, as well as the ability of radicals to interact with water (hydrate). TO hydrophilic include radicals containing cationic, anionic and polar uncharged functional groups. TO hydrophobic– radicals containing alkyl or aryl groups.
Depending on polarity R-groups there are four classes of amino acids: nonpolar, polar uncharged, negatively charged and positively charged.
Non-polar amino acids include: glycine; amino acids with alkyl and aryl side chains - alanine, valine, leucine, isoleucine; tyrosine, tryptophan, phenylalanine; imino acid - proline. They strive to get into the hydrophobic environment “inside” the protein molecule (Fig. 1).
Rice. 1. Non-polar amino acids
Polar charged amino acids include: positively charged amino acids – histidine, lysine, arginine (Fig. 2); negatively charged amino acids - aspartic and glutamic acid(Fig. 3). They usually protrude outward into the protein's aqueous environment.
The remaining amino acids form the category of polar uncharged: serine and threonine (amino acids-alcohols); asparagine and glutamine (amides of aspartic and glutamic acids); cysteine and methionine (sulfur-containing amino acids).
Since at neutral pH the COOH groups of glutamic and aspartic acids are completely dissociated, they are usually called glutamate And aspartate regardless of the nature of the cations present in the medium.
A number of proteins contain special amino acids that are formed by modifying ordinary amino acids after their inclusion in the polypeptide chain, for example, 4-hydroxyproline, phosphoserine, -carboxyglutamic acid, etc. 
Rice. 2. Amino acids with charged side groups
All amino acids formed during the hydrolysis of proteins under fairly mild conditions exhibit optical activity, i.e., the ability to rotate the plane of polarized light (with the exception of glycine).
Rice. 3. Amino acids with charged side groups
All compounds that can exist in two stereoisomeric forms, L- and D-isomers, have optical activity (Fig. 4). Proteins contain only L-amino acids. 
L-alanine D-alanine
Rice. 4. Optical isomers of alanine
Glycine has no asymmetric carbon atom, while threonine and isoleucine each contain two asymmetric carbon atoms. All other amino acids have one asymmetric carbon atom.
The optically inactive form of an amino acid is called a racemate, which is an equimolar mixture D- And L-isomers, and is designated by the symbol D.L.-.
M 
The amino acid numbers that make up polypeptides are called amino acid residues. Amino acid residues are connected to each other by a peptide bond (Fig. 5), in the formation of which the α-carboxyl group of one amino acid and the α-amino group of another take part.
Rice. 5. Peptide bond formation
The equilibrium of this reaction is shifted towards the formation of free amino acids rather than the peptide. Therefore, the biosynthesis of polypeptides requires catalysis and energy expenditure.
Since the dipeptide contains a reactive carboxyl and amino group, other amino acid residues can be attached to it with the help of new peptide bonds, resulting in the formation of a polypeptide - a protein.
The polypeptide chain consists of regularly repeating sections - NHCHRCO groups, forming the main chain (skeleton or backbone of the molecule), and a variable part, including characteristic side chains. R- groups of amino acid residues protrude from the peptide backbone and largely form the surface of the polymer, determining many physical and Chemical properties proteins. Free rotation in the peptide backbone is possible between the nitrogen atom of the peptide group and the neighboring α-carbon atom, as well as between the α-carbon atom and the carbon of the carbonyl group. Due to this, the linear structure can acquire a more complex spatial conformation.
An amino acid residue containing a free α-amino group is called N-terminal, and having a free -carboxyl group – WITH-end.
The structure of peptides is usually depicted with N-end.
Sometimes the terminal -amino and -carboxyl groups bind to each other, forming cyclic peptides.
Peptides differ in the number of amino acids, amino acid composition and the order of amino acid connection.
Peptide bonds are very strong, and their chemical hydrolysis requires harsh conditions: high temperature and pressure, an acidic environment and a long time.
In a living cell, peptide bonds can be broken by proteolytic enzymes called proteases, or peptide hydrolases.
Just like amino acids, proteins are amphoteric compounds and are charged in aqueous solutions. Each protein has its own isoelectric point - the pH value at which the positive and negative charges of the protein are completely compensated and the total charge of the molecule is zero. At pH values above the isoelectric point, the protein carries a negative charge, and at pH values below the isoelectric point, it carries a positive charge.
SEQUENATORS. STRATEGY AND TACTICS OF PRIMARY STRUCTURE ANALYSIS
Determining the primary structure of proteins comes down to determining the order of amino acids in the polypeptide chain. This problem is solved using the method sequencing(from English sequence-subsequence).
In principle, the primary structure of proteins can be determined by direct analysis amino acid sequence or by deciphering the nucleotide sequence of the corresponding genes using the genetic code. Naturally, the greatest reliability is ensured by a combination of these methods.
Sequencing itself at its current level makes it possible to determine the amino acid sequence in polypeptides whose size does not exceed several tens of amino acid residues. At the same time, the polypeptide fragments under study are much shorter than those natural proteins with which we have to deal. Therefore, preliminary cutting of the original polypeptide into short fragments is necessary. After sequencing the resulting fragments, they must be stitched back together in the original sequence.
Thus, determining the primary sequence of a protein comes down to the following main steps:
1) cleavage of the protein into several fragments of length accessible for sequencing;
2) sequencing of each of the obtained fragments;
3) assembly of the complete protein structure from the established structures of its fragments.
The study of the primary structure of a protein consists of the following stages:
– determination of its molecular weight;
– determination of specific amino acid composition (AA composition);
- definition N- And WITH-terminal amino acid residues;
– splitting of the polypeptide chain into fragments;
– cleavage of the original polypeptide chain in another way;
– separation of the resulting fragments;
– amino acid analysis of each fragment;
– establishment of the primary structure of the polypeptide, taking into account the overlapping sequences of fragments of both cleavages.
Since there is no method yet that allows one to establish the complete primary structure of a protein on an entire molecule, the polypeptide chain is subjected to specific cleavage with chemical reagents or proteolytic enzymes. The mixture of the resulting peptide fragments is separated and the amino acid composition and amino acid sequence are determined for each of them. After the structure of all fragments has been established, it is necessary to determine the order of their location in the original polypeptide chain. To do this, the protein is subjected to cleavage using another agent and a second, different set of peptide fragments is obtained, which are separated and analyzed in a similar way.
1. Determination of molecular weight (the following methods are discussed in detail in topic 3):
– by viscosity;
– by sedimentation rate (ultracentrifugation method);
– gel chromatography;
– electrophoresis in PAGE under dissociating conditions.
2. Determination of AA composition. Analysis of amino acid composition includes complete acid hydrolysis of the protein or peptide under study using 6 n. of hydrochloric acid and quantification of all amino acids in the hydrolyzate. Hydrolysis of the sample is carried out in sealed ampoules in a vacuum at 150°C for 6 hours. Quantitative determination of amino acids in a protein or peptide hydrolyzate is carried out using an amino acid analyzer.
3. Determination of N- and C-amino acid residues. In the polypeptide chain of a protein, on one side there is an amino acid residue carrying a free α-amino group (amino or N-terminal residue), and on the other - a residue with a free α-carboxyl group (carboxyl, or WITH-terminal residue). Analysis of terminal residues plays an important role in the process of determining the amino acid sequence of a protein. At the first stage of the study, it makes it possible to estimate the number of polypeptide chains that make up the protein molecule and the degree of homogeneity of the drug under study. In subsequent stages, using analysis N-terminal amino acid residues control the process of separation of peptide fragments.
Reactions for determining N-terminal amino acid residues:
1) one of the first methods for determining N-terminal amino acid residues was proposed by F. Sanger in 1945. When the α-amino group of a peptide or protein reacts with 2,4-dinitrofluorobenzene, a dinitrophenyl (DNP) derivative is obtained, colored yellow. Subsequent acid hydrolysis (5.7 N HCl) leads to cleavage of peptide bonds and the formation of a DNP derivative N-terminal amino acid. The DNP amino acid is extracted with ether and identified by chromatography in the presence of standards.
2) dansylation method. Greatest application for determining N-terminal residues are currently found by the dansil method, developed in 1963 by W. Gray and B. Hartley. Like the dinitrophenylation method, it is based on the introduction of a “tag” into the amino groups of the protein, which is not removed during subsequent hydrolysis. Its first step is the reaction of dansyl chloride (1-dimethylaminonaphthalene-5-sulfochloride) with the unprotonated α-amino group of a peptide or protein to form dansyl peptide (DNS peptide). At the next stage, the DNS peptide is hydrolyzed (5.7 N HC1, 105°C, 12 - 16 h) and released N-terminal α-DNS amino acid. DNS amino acids exhibit intense fluorescence in the ultraviolet region of the spectrum (365 nm); Usually 0.1 - 0.5 nmol of the substance is sufficient for their identification.
There are a number of methods that can be used to determine how N-terminal amino acid residue and amino acid sequence. These include degradation by the Edman method and enzymatic hydrolysis by aminopeptidases. These methods will be discussed in detail below when describing the amino acid sequence of peptides.
Reactions for determining C-terminal amino acid residues:
1) among chemical methods of determination WITH-terminal amino acid residues, the hydrazinolysis method proposed by S. Akabori and the oxazolone method deserve attention. In the first of them, when a peptide or protein is heated with anhydrous hydrazine at 100 - 120°C, the peptide bonds are hydrolyzed to form amino acid hydrazides. WITH The -terminal amino acid remains as a free amino acid and can be isolated from the reaction mixture and identified (Fig. 6).
Rice. 6. Cleavage of the peptide bond with hydrazine
The method has a number of limitations. Hydrazinolysis destroys glutamine, asparagine, cysteine and cystine; arginine loses its guanidine moiety to form ornithine. Serine, threonine, and glycine hydrazides are labile and easily converted into free amino acids, making the results difficult to interpret;
2) The oxazolone method, often called the tritium tag method, is based on the ability WITH-terminal amino acid residue undergoes cyclization under the influence of acetic anhydride to form oxazolone. Under alkaline conditions, the mobility of hydrogen atoms at position 4 of the oxazolone ring sharply increases and they can be easily replaced by tritium. The reaction products formed as a result of subsequent acid hydrolysis of the tritiated peptide or protein contain radioactively labeled WITH-terminal amino acid. Chromatography of the hydrolyzate and measurement of radioactivity allows identification WITH-terminal amino acid of a peptide or protein;
3) most often to determine WITH-terminal amino acid residues are enzymatically hydrolyzed by carboxypeptidases, which also allows the C-terminal amino acid sequence to be analyzed. Carboxypeptidase hydrolyzes only those peptide bonds that are formed WITH-terminal amino acid having a free α-carboxyl group. Therefore, under the action of this enzyme, amino acids are sequentially cleaved from the peptide, starting with WITH-terminal. This allows you to determine mutual arrangement alternating amino acid residues.
As a result of identification N- And WITH-terminal residues of the polypeptide provide two important reference points for determining its amino acid sequence (primary structure).
4. Fragmentation of the polypeptide chain.
Enzymatic methods. For specific breakdown of proteins at certain points, both enzymatic and chemical methods are used. Of the enzymes that catalyze the hydrolysis of proteins at specific points, trypsin and chymotrypsin are the most widely used. Trypsin catalyzes the hydrolysis of peptide bonds located after lysine and arginine residues. Chymotrypsin preferentially breaks down proteins after aromatic amino acid residues - phenylalanine, tyrosine and tryptophan. If necessary, the specificity of trypsin can be increased or changed. For example, treatment of the protein under study with citraconic anhydride leads to acylation of lysine residues. In such a modified protein, cleavage will occur only at arginine residues. Also when studying the primary structure of proteins wide application finds a proteinase, which also belongs to the class of serine proteinases. The enzyme has two maxima of proteolytic activity at pH 4.0 and 7.8. Proteinase cleaves peptide bonds formed by the carboxyl group of glutamic acid with high yield.
Researchers also have at their disposal a large set of less specific proteolytic enzymes (pepsin, elastase, subtilisin, papain, pronase, etc.). These enzymes are mainly used for additional fragmentation of peptides. Their substrate specificity is determined by the nature of amino acid residues, not only forming a hydrolyzable bond, but also more distant along the chain.
Chemical methods.
1) among the chemical methods of protein fragmentation, the most specific and most often used is cyanogen bromide cleavage at methionine residues (Figure 7).
The reaction with cyanogen bromide results in the formation of the intermediate cyanosulfonium derivative of methionine, which spontaneously converts under acidic conditions to homoserine iminolactone, which, in turn, is rapidly hydrolyzed with cleavage of the imine bond. Resulting on WITH-terminus of the peptides, the homoserine lactone is further partially hydrolyzed to homoserine (HSer), resulting in each peptide fragment except WITH-terminal, exists in two forms - homoserine and homoserine lactone; 
Rice. 7. Cleavage of the polypeptide chain with cyanogen bromide
2) a large number of methods have been proposed for protein cleavage at the carbonyl group of the tryptophan residue. One of the reagents used for this purpose is N-bromosuccinimide;
3) thiol-disulfide exchange reaction. Reduced glutathione, 2-mercaptoethanol, and dithiothreitol are used as reagents.
5. Determination of the sequence of peptide fragments. At this stage, the amino acid sequence in each of the peptide fragments obtained in the previous stage is established. For this purpose they usually use chemical method, designed by Per Edman. Edman cleavage boils down to the fact that only N-terminal residue of the peptide, and all other peptide bonds are not affected. After identifying the split-off N- the terminal remainder of the label is introduced into the next one, which has now become N-terminal, a residue that is cleaved off in the same way, going through the same series of reactions. Thus, by eliminating residue by residue, it is possible to determine the entire amino acid sequence of a peptide using just one sample for this purpose. In the Edman method, the peptide first reacts with phenyl isothiocyanate, which attaches to the free α-amino group N-terminal residue. Treatment of the peptide with cold dilute acid leads to elimination N-terminal residue in the form of a phenylthiohydantoin derivative, which can be identified by chromatographic methods. The rest of the peptide value after removal N-terminal residue appears intact. The operation is repeated as many times as there are residues in the peptide. In this way, the amino acid sequence of peptides containing 10 - 20 amino acid residues can be easily determined. The amino acid sequence is determined for all fragments formed during cleavage. After this, the next problem arises - to determine in what order the fragments were located in the original polypeptide chain.
Automatic determination of amino acid sequence . A major achievement in the field of structural studies of proteins was the creation in 1967 by P. Edman and J. Begg sequencer– a device that carries out sequential automatic elimination with high efficiency N-terminal amino acid residues using the Edman method. Modern sequencers implement various methods determining the amino acid sequence.
6. Cleavage of the original polypeptide chain in another way. To establish the order of arrangement of the resulting peptide fragments, take a new portion of the original polypeptide preparation and split it into smaller fragments in some other way, by which peptide bonds that are resistant to the action of the previous reagent are cleaved. Each of the resulting short peptides is subjected to sequential cleavage using the Edman method (the same as in the previous stage), and in this way their amino acid sequence is determined.
7. Establishment of the primary structure of the polypeptide, taking into account the overlapping sequences of fragments of both cleavages. The amino acid sequences in the peptide fragments obtained by the two methods are compared to find peptides in the second set in which the sequences of individual sections would match the sequences of certain sections of the peptides of the first set. Peptides from the second set with overlapping regions allow the peptide fragments obtained as a result of the first cleavage of the original polypeptide chain to be connected in the correct order.
Sometimes a second cleavage of a polypeptide into fragments is not enough to find overlapping regions for all peptides obtained after the first cleavage. In this case, a third, and sometimes a fourth, cleavage method is used to obtain a set of peptides that ensure complete overlap of all regions and establish the complete amino acid sequence in the original polypeptide chain.
The word “supplements” has recently become almost a dirty word among some doctors. Meanwhile, dietary supplements are not at all useless and can bring tangible benefits. The disdainful attitude towards them and the loss of trust among people is due to the fact that many falsifications have appeared on the crest of the craze for biologically active substances. Since our site often talks about preventive measures, helping to maintain health, it is worth touching on this issue in more detail - what refers to biologically active substances and where to look for them.
What are biologically active substances?
Biologically active substances mean substances that have high physiological activity and affect the body in the smallest doses. They can accelerate metabolic processes, improve metabolism, participate in the synthesis of vitamins, and help regulate the proper functioning of body systems.
BAVs can play different roles. A number of similar substances, when studied in detail, have shown their ability to suppress growth cancerous tumors. Other substances such as ascorbic acid, participate in a huge number processes occurring in the body and help strengthen the immune system.
Dietary supplements, or dietary supplements, are preparations based on an increased concentration of certain biologically active substances. They are not considered a medicine, but they can successfully treat diseases associated with an imbalance of substances in the body.
As a rule, biologically active substances are found in plants and animal products, so many drugs are made based on them.
Types of biologically active substances
The therapeutic effect of herbal medicine and various dietary supplements is explained by the combination of active substances contained. What substances are considered biologically active by modern medicine? These are well-known vitamins, fatty acids, micro- and macroelements, organic acids, glycosides, alkaloids, phytoncides, enzymes, amino acids and a number of others. We have already written about the role of microelements in the article, now let’s talk more specifically about other biologically active substances.
Amino acids
From the school biology course we know that amino acids are part of proteins, enzymes, many vitamins and other organic compounds. IN human body 12 of the 20 essential amino acids are synthesized, that is, there are a number of essential amino acids that we can only get from food.
Amino acids serve for the synthesis of proteins, which in turn form glands, muscles, tendons, hair - in a word, all parts of the body. Without certain amino acids, the normal functioning of the brain is impossible, since it is the amino acid that allows the transmission of nerve impulses from one nerve cell to another. In addition, amino acids regulate energy metabolism and ensure that vitamins and microelements are absorbed and work fully.
The most important amino acids include tryptophan, methionine and lysine, which are not synthesized by humans and must be supplied with food. If there are not enough of them, then you need to take them as part of a dietary supplement.
Tryptophan is found in meat, bananas, oats, dates, sesame seeds, and peanuts; methionine - in fish, dairy products, eggs; lysine - in meat, fish, dairy products, wheat.
If there are not enough amino acids, the body tries to extract them first from its own tissues. And this leads to their damage. First of all, the body extracts amino acids from the muscles - it is more important for it to feed the brain than the biceps. Hence, the first symptom of a lack of essential amino acids is weakness, fast fatiguability, exhaustion, then anemia, loss of appetite and deterioration of skin condition join this.
A lack of essential amino acids in childhood is very dangerous - this can lead to delayed growth and mental development.
Carbohydrates
Everyone has heard about carbohydrates from glossy magazines - women losing weight consider them their number one enemy. Meanwhile, carbohydrates play vital role in the construction of body tissues and their lack leads to sad consequences - low-carb diets demonstrate this constantly.
Carbohydrates include monosaccharides (glucose, fructose), oligosaccharides (sucrose, maltose, stachyose), polysaccharides (starch, fiber, inulin, pectin, etc.).
Fiber acts as a natural detoxifier. Inulin reduces cholesterol and sugar levels in the blood, helps increase bone density, and strengthens the immune system. Pectin has an antitoxic effect, lowers cholesterol levels, has a beneficial effect on the cardiovascular system and strengthens the immune system. Pectin is found in apples, berries, and many fruits. There is a lot of inulin in chicory and Jerusalem artichoke. Vegetables and grains are rich in fiber. Bran is most often used as an effective dietary supplement containing fiber.
Glucose is essential for proper brain function. It is found in fruits and vegetables.
Organic acids
Organic acids support the body acid-base balance and participate in many metabolic processes. Each acid has its own spectrum of action. Ascorbic and succinic acids have a powerful antioxidant effect, for which they are also called the elixir of youth. Benzoic acid has an antiseptic effect and helps fight inflammatory processes. Oleic acid improves the functioning of the heart muscle and prevents muscle atrophy. A number of acids are part of hormones.
Many organic acids are found in vegetables and fruits. You should be aware that consuming too many dietary supplements containing organic acids can lead to a disservice being provided to the body - the body will become excessively alkalized, which will lead to disruption of the liver and a deterioration in the removal of toxins.
Fatty acid
The body can synthesize many fatty acids on its own. It cannot produce only polyunsaturated acids, which are called omega-3 and 6. About the benefits of unsaturated fatty acids Only the lazy have not heard of omega-3 and omega-6.
Although they were discovered at the beginning of the 20th century, their role began to be studied only in the 70s of the last century. Nutritionists have found that people who eat fish rarely suffer from hypertension and atherosclerosis. Since fish is rich in omega-3 acids, people quickly became interested in them. It turned out that omega-3 has a beneficial effect on joints, blood vessels, blood composition, and skin condition. It was found that this acid restores hormonal balance and also allows you to regulate calcium levels - today it is successfully used for the treatment and prevention of early aging, Alzheimer's disease, migraines, osteoprosis, diabetes mellitus, hypertension, atherosclerosis.
Omega-6 helps regulate the functioning of the hormonal system, improve the condition of the skin and joints, especially in cases of arthritis. Omega-9 is an excellent cancer preventative.
A lot of omega-6 and 9 are found in lard, nuts, and seeds. Omega-3 is found, in addition to fish and seafood, in vegetable oils, fish oil, eggs, legumes.
Resins
Surprisingly, they are also biologically active substances. They are found in many plants and have valuable medicinal properties. Thus, the resins contained in birch buds have an antiseptic effect, and the resins of coniferous trees have an anti-inflammatory, anti-sclerotic, and wound-healing effect. Especially a lot useful properties in resin used to prepare fir and cedar balsams.
Phytoncides
Phytoncides have the ability to destroy or inhibit the proliferation of bacteria, microorganisms, and fungi. It is known that they kill the influenza virus, dysentery and tuberculosis bacillus, have a wound-healing effect, and regulate secretory function gastrointestinal tract, improve cardiac activity. The phytoncidal properties of garlic, onions, pine, spruce, and eucalyptus are especially valued.
Enzymes
Enzymes are biological catalysts for many processes occurring in the body. They are sometimes called enzymes. They help improve digestion, remove toxins from the body, stimulate brain activity, strengthen the immune system, participate in the renewal of the body. May be of plant or animal origin.
Recent research clearly states that in order for plant enzymes to work, the plant must not be cooked before eating. Cooking kills enzymes and renders them useless.
Particularly important for the body is coenzyme Q10, a vitamin-like compound that is normally produced in the liver. It is a powerful catalyst for a number of vital processes, especially the formation of the ATP-o molecule, an energy source. Over the years, the process of coenzyme production slows down, and in old age there is very little of it. It is believed that a lack of coenzyme is to blame for aging.
Today it is proposed to introduce coenzyme Q10 into the diet artificially with dietary supplements. Such drugs are widely used to improve heart function, improve appearance skin, improved performance immune system, in order to combat excess weight. We once wrote about, here we will add that when taking coenzyme, you should take these recommendations into account too.
Glycosides
Glycosides are compounds of glucose and other sugars with a non-sugar part. Cardiac glycosides contained in plants are useful for heart diseases and normalize its functioning. Such glycosides are found in digitalis, lily of the valley, and jaundice.
Anthraglycosides have a laxative effect and are also capable of dissolving kidney stones. Anthraglycosides are found in buckthorn bark, rhubarb roots, horse sorrel, and madder.
Saponins have different effects. Thus, horsetail saponins have a diuretic effect, licorice has an expectorant effect, ginseng and aralia have a tonic effect.
There are also bitters that stimulate the secretion of gastric juice and normalize digestion. Interestingly, their chemical structure has not yet been studied. Bitterness is contained in wormwood.
Flavonoids
Flavonoids are phenolic compounds found in many plants. By therapeutic effect flavonoids are similar to vitamin P - rutin. Flavonoids have vasodilating, anti-inflammatory, choleretic, and vascular strengthening properties.
Tannins are also classified as phenolic compounds. These biologically active substances have a hemostatic, astringent and antimicrobial effect. These substances contain oak bark, burnet, lingonberry leaves, bergenia root, and alder cones.
Alkaloids
Alkaloids are biologically active nitrogen-containing substances found in plants. They are very active, most alkaloids in high dose poisonous. In a small space this is the most valuable remedy. As a rule, alkaloids have a selective effect. Alkaloids include substances such as caffeine, atropine, quinine, codeine, and theobromine. Caffeine has a stimulating effect on the nervous system, and codeine, for example, suppresses cough.
Knowing what biologically active substances are and how they work, you can choose dietary supplements more intelligently. This, in turn, will allow you to select exactly the drug that will really help you cope with health problems and improve your quality of life.
