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

How often do we feel a deficiency of vitamins and minerals almost every year due to the piled-up melancholy and fatigue, and willingly, out of habit, we attribute it to “avitaminosis”. But it is important to understand that many health problems can be associated with a lack of quality protein. And this, unfortunately, we very rarely worry 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:

Cravings for sweets

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 with the restriction of protein foods, we are in no hurry to lean on meat and eggs - the main task of proteins is to maintain blood sugar levels. And it is sweets that help to quickly correct the situation.

Poor concentration

The concentration will be excellent only with a balanced blood sugar level. And when this level is subject to constant fluctuations, then it may well be a feeling of foggy consciousness, 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 indispensable 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 well known that proteins are 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 human immune system directly depends on the systematic influx of protein. That is why quite frequent colds and infectious diseases- a clear indication of a lack of proteins.

What contains protein

Animal and vegetable proteins

Most plant foods contain no less protein than milk or chicken. But the human body is arranged in such a way that, as usual, protein is partially absorbed, everything else is excreted with urine. You should consume protein of both plant and animal origin - but this is ideal. If you're into any type of vegetarian diet, you'll just need to balance your diet to make up 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;
• chicken.

All of these products contain both protein and fat, but not in the least amount. It should not be forgotten that from products containing protein, it is recommended to give preference to dairy products, the fat content of which is not more than 3%, skinless chicken and lean meat. As for cheeses, then fat content is allowed up to 40%.

Vegetable protein

Since vegetarianism is in fashion 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.

Plant protein is digestible from cereals, however, you need to know, at least to combine with animal protein, in which cereals the protein is contained in large quantities:

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

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

Beauty directly depends on health, and much knowledge about the effect of certain substances on our appearance we have absorbed from childhood. So for nails, for example, not only calcium is important, but also protein. Therefore, if you want your manicure to be exceptionally admirable, you should think about whether there is a lack of protein in your diet. Also on how much dietary protein in your plate, the condition of your hair will also depend, so perhaps you should take the issue seriously.

Where's the cake?

We eat cake...

Pigs, we need another cake!

We also eat others...

Pigs, cupcakes left?

Eat too...

There are lollipops!

I will eat them!

Need to share?!

And you share croissants with me?!

Shrek 4 (Shrek Forever After)

Lack of protein in the body

Proteins are one of the most essential substances in the human body. If we remember the deficiency of vitamins and minerals almost every spring, attributing the blues and fatigue to “avitaminosis”, then we think little about the fact that many health problems can be associated with a deficiency of high-quality protein.

Many say that protein is a heavy product, and it should be eaten in a limited way. And some do not eat it at all - and it seems that nothing bad is happening. However, protein in the body performs vital functions that no other element can take on. What is the purpose of protein in the human body?

Why are proteins needed?

Protein is the foundation for building the body. Proteins make up muscles, tissues, internal organs, blood cells, immune bodies, as well as hair, nails and skin cells and proteins.

Dietary proteins in the body in the intestines are disassembled to "bricks" of amino acids. Amino acids are sent to the liver to build and synthesize the body's own proteins, but there are some amino acids in the body that the body can produce itself, and some must only come from outside. These are essential acids, but only animal proteins contain them; in plant proteins, the set of amino acids is poorer, so they are not considered complete.

Another important function of the protein is its enzymatic and metabolic function. Most enzymes and hormones are pure protein or a combination of protein with other substances (metal ions, fats, vitamins). With a lack of protein, some types of metabolism can suffer, this is especially noticeable with restrictive low-protein diets.

In addition, proteins perform a transport function, that is, they carry important substances into and out of cells - ions, nutrients and other substances. Proteins protect our body from infections, since antibodies and protective mucosal proteins are protein molecules.

Proteins support our youth and beauty - and this is due to the timely renewal of collagen and elastin molecules, which prevent dehydration, aging of our skin, and prevent the formation of wrinkles.

How to determine if you have a protein deficiency?

1.Look at yourself in the mirror. If you have flabby muscles, saggy skin, wrinkles on your face, and you are not yet thirty, you definitely have problems with protein metabolism. If you are actively training, while consuming almost no protein, fasting or low-protein diets, you also have problems with protein metabolism. You should review your diet if your weight is more than 25% above normal, and even more so if you are obese. With a lack of protein, the metabolism slows down, which reduces the activity of enzymes and hormones, and this ultimately leads to a loss muscle mass and a set of fat instead.

2. Consider your skin, nails and hair, what is their condition? They are almost entirely of protein origin, and when it is deficient, they suffer seriously. If the body lives in conditions of chronic protein deficiency, flabbiness and pallor of the skin, its defects, brittle hair, exfoliating and poorly growing nails appear.

3. Immunity problems - frequent colds, allergies, dermatitis and pustular rashes. Basically, they are also associated with protein deficiency, immune cells and antibodies simply have nothing to build from.

4. There may be digestive disorders, constipation, general malaise, fatigue, low resistance to stress.

How to replenish protein stores

In order to avoid problems with protein starvation and disorders associated with a lack of protein in the body, it is necessary to take a number of preventive measures, primarily related to nutrition and lifestyle.

1. Critically review your diet

You may think that you eat a lot of meat, but in fact, these products contain very little quality food protein (or not at all). Relatively classified as meat and meat products are such traditional products of our table as:

Boiled or smoked sausages, sausages and sausages, even made in accordance with GOST. Protein in them is critically small for the full provision of the body.

Semi-finished products with "meat", store cutlets, dumplings. The role of meat there is played by soy proteins and flavorings.

Smoked hams, shanks, rolls, etc. The meat there undergoes thermal or marinade treatments, its quality also suffers. It is also unknown where, how and from what meat it was prepared, whether elementary sanitary standards were observed.

You can occasionally enjoy these foods as a variety, but you should not use them often - especially as a source of protein!

2. Choose lean meats and lean fish

Fats interfere with the full absorption of protein. The fattest are salmon, catfish, goose and duck, cod liver, pork and beef brisket. The best sources of protein are skinless chicken, beef, rabbit, turkey, and eggs, although plant-based protein in the form of legumes, nuts, and buckwheat should also be added to your diet.

At the same time, the most useful ways of cooking meat are baking in foil, grilling, barbecue, steaming, stewing. Roasting meat is the most harmful cooking method.

3. Eat Protein Separately

Protein foods should be eaten separately from potatoes, cereals and without bread, as they are poorly digested. It is better to combine meat with vegetables - fresh or stewed, they will help to absorb protein. Eat protein foods before 6 pm, as digestion is difficult at night.

At the same time, you should not overload the body with protein, since an excess of protein leads to rotting processes in the intestines and intoxication with metabolic products, constipation and a feeling of heaviness in the stomach.

No. 1. Proteins: peptide bond, their detection.

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

Squirrels are macromolecular compounds built from amino acids. 20 amino acids are involved in making proteins. They link together into long chains that form the backbone of a large molecular weight protein molecule.

Functions of proteins in the body

The combination of peculiar chemical and physical properties of proteins provides this particular 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. To date, thousands of enzymes have been characterized, many of them isolated in crystalline form. Almost all enzymes are powerful catalysts, increasing the rates of reactions by at least a million times. This function of proteins is unique, not characteristic of other polymeric 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. Many small molecules and ions are transported by specific proteins. For example, the respiratory function of blood, namely the transport of oxygen, is performed by hemoglobin molecules, a protein in red blood cells. Serum albumins are 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 provides 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. The coagulation of a blood plasma protein - fibrinogen - and the formation of a blood clot that protects against blood loss during injuries is another example of the protective function of proteins.

5. Contractile function of proteins. Many proteins are involved in the act of muscle contraction and relaxation. The main role in these processes is played by actin and myosin - specific proteins of muscle tissue. The contractile function is also inherent in the proteins of subcellular structures, which provides the finest processes of cell vital activity,

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

7. Hormonal (regulatory) function of proteins. Metabolism in the body is regulated by various mechanisms. In this regulation, an important place is occupied by hormones produced by 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), whose molecules are built from amino acid residues. Protein monomers are α-amino acids, a 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's ideas about the role of peptide bonds -CO-NH- in the construction of a protein molecule, the German scientist E. Fischer proposed at the beginning of the 20th century the peptide theory of the structure of proteins. According to this theory, proteins are linear polymers of α-amino acids linked by a peptide bond - 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). The structure of peptides is usually depicted 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 denotes a peptide in which the N-terminal α-amino acid is ­ lyatsya alanine, and the C-terminal - cysteine. When reading such a record, the endings of the names of all acids, except for the last ones, change to - "yl": alanyl-tyrosyl-leucyl-seryl-tyrosyl--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, when hydrolyzed, give only amino acids.

Proteinoids ____simple proteins of animal origin, insoluble in water, salt solutions, dilute acids and alkalis. They perform mainly supporting functions (for example, collagen, keratin

protamines - positively charged nuclear proteins, with a molecular weight of 10-12 kDa. Approximately 80% are composed of alkaline amino acids, which makes it possible for them 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 relate to 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, chemical structure features - a lot of proline, glutamic acid, no lysine ,

glutelins - soluble in alkaline solutions ,

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

albumins - proteins of animal and plant tissues, soluble in water and saline solutions. The molecular weight is 69 kDa;

scleroproteins - proteins of the supporting tissues of animals

Examples of simple proteins are silk fibroin, egg serum albumin, pepsin, etc.

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

No. 4. Proteins as polyelectrolytes. Isoelectric point of a 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 a protein solution is acidified, the degree of ionization of anionic groups decreases, while that of cationic groups increases; when alkalized - vice versa. At a certain pH value, the number of positively and negatively charged groups becomes the same, and the isoelectric state of the protein appears (the total charge is 0). The pH value at which the 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 milt (pl=12). In addition, there are proteins that have a very low pI value, for example, pepsin, an enzyme of gastric juice (pl=l). At the isoelectric point, proteins are very unstable and precipitate easily, having the least solubility.

If the protein is not in an isoelectric state, then electric field its molecules will move towards the cathode or anode, depending on the sign of the total charge and at a speed 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 buffer properties in the pH range of 6-8. There are very few of these proteins. For example, hemoglobin, containing almost 8% histidine, is a powerful intracellular buffer in red blood cells, maintaining the pH of the blood at a constant level.

No. 5. Physico-chemical 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 reactions of nitration, acylation, alkylation, esterification, redox and others. Proteins have acid-base, buffer, colloidal and osmotic properties.

Acid-base properties of proteins

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

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

Physico-chemical 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 a lot of glu and asp, which have additional carboxyl and fewer amino groups. There are many lys and args in alkaline proteins. Each protein molecule in an aqueous solution is surrounded by a hydration shell, since proteins have many hydrophilic groups (-COOH, -OH, -NH 2, -SH) due to amino acids. In aqueous solutions, the protein molecule has a charge. The charge of protein in water can change depending on the pH.

Protein precipitation. Proteins have a hydration shell, a charge that prevents sticking. For deposition, it is necessary to remove the hydrate 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. 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 the protein globules, the hydrate (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 some organic solvents, such as 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 of 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.

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

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

To detect proteins in solution, the following are used:

color reactions;

precipitation reactions.

Methods for isolation and purification of proteins.

homogenization- the 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 - spatial structure squirrel. This is either a spiral or a folding. Hydrogen bonds are created.

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

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

Example: hemoglobin \u003d -globule + -globule - carries O 2 in the aggregate, and not separately.

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

6) Methods for detecting proteins.

Proteins are high-molecular biological polymers, the structural (monomeric) units of which are -amino acids. Amino acids in proteins are linked to each other by peptide bonds. the formation of which occurs due to the carboxyl group standing at -carbon atom of one amino acid and -amine group of another amino acid with the release of a water molecule. The 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. Chemical and physical properties proteins are due to the chemical nature and physicochemical properties of the radicals, their constituent amino acid residues. Methods for the detection and quantification of proteins in biological objects and food products, as well as their isolation from tissues and biological fluids, are based on physical and chemical properties 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 allow you to set the presence of a protein in a biological object or solution and prove the presence certain amino acids in a protein molecule. On the basis of color reactions, some methods for the quantitative determination of proteins and amino acids have been developed.

Consider universal biuret and ninhydrin reactions, since all proteins give them. Xantoprotein reaction, Fohl reaction and others are specific, since they are due to the radical groups of certain amino acids in the protein molecule.

Color reactions allow you to establish the presence of a protein in the material under study 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 medium form with copper(II) ions complex compounds colored in purple (with a red or blue tinge) color. The color 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 =C─O ˉ groups and four coordination bonds with nitrogen atoms (=N−).

The color intensity depends on the amount of protein in the solution. This makes it possible to use this reaction for the quantitative determination of protein. 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 given 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. The color in this reaction develops due to the α-amino group.

-amino acids react very easily with ninhydrin. Along with them, Rueman'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 to detect and quantify amino acids.

xantoprotein reaction. This reaction indicates the presence of aromatic amino acid residues in proteins - tyrosine, phenylalanine, tryptophan. It is based on the nitration of the benzene ring of the radicals of these amino acids with the formation of yellow-colored nitro compounds (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 the 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).

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

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

The transition of sulfur 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 ions and lead, black or brown lead sulfide is formed:

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

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

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

Millon 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 reaction for histidine and tyrosine . The Pauli reaction makes it possible to detect the amino acids histidine and tyrosine in the protein, which form cherry-red complex compounds with diazobenzenesulfonic acid. Diazobenzenesulfonic acid is formed in the reaction of diazotization when sulfanilic acid reacts 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 sodium nitrite solution are added to the test solution, mixed thoroughly and soda (sodium carbonate) is immediately added. After stirring, the mixture turns cherry red, provided that histidine or tyrosine is present in the test solution.

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

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

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 gently 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 evenly turn purple.

TO

condensation 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.

Strict nomenclature and classification of proteins still does not exist. The names of proteins are given randomly, 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.

Proteins are classified according to composition, particle shape, solubility, amino acid composition, origin, etc.

1. Composition Proteins are divided into two large groups: simple and complex proteins.

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

Complex (proteids) 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 in their composition metals (Fe, Cu, Mg, etc.) associated directly with the polypeptide chain;

Phosphoproteins - contain residues of phosphoric acid, which are attached to the protein molecule by ester bonds 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 may contain derivatives of porphyrin, isoalloxazine, and carotene;

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

Nucleoproteins are proteins that consist of a single protein and a nucleic acid linked 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. Particle shape- proteins are divided into fibrillar (thread-like) 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 - proteins of supporting tissues (bones, cartilage, ligaments, tendons, hair, nails, skin, etc.). These are mainly fibrillar proteins with a large 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) - proteins that are soluble in water and contain 80-90% arginine and a limited set (6-8) of other amino acids, are present in the milk of various fish. Due to the high content of arginine, they have basic properties, their molecular weight is relatively small and is approximately equal to 4000-12000 Da. They are a protein component in the composition of nucleoproteins;

Histones are highly soluble in water and dilute acid solutions (0.1 N), have a high content of amino acids: arginine, lysine and histidine (at least 30%) and therefore have basic properties. These proteins are found in significant amounts 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 saline solutions with a salt concentration of more than 7%. Globulins are completely precipitated at 50% saturation of the solution with ammonium sulfate. These proteins are characterized by a high content of glycine (3.5%), their molecular weight > 100,000 Da. Globulins are weakly acidic or neutral proteins (p1=6-7.3);

Albumins are proteins that are highly soluble in water and strong saline solutions, and the salt concentration (NH 4) 2 S0 4 should not exceed 50% of saturation. At higher concentrations, albumins are salted out. Compared to globulins, these proteins contain three times less glycine and have a molecular weight of 40,000-70,000 Da. Albumins have an excess 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 cereals. 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 - vegetable proteins are 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 contain more arginine and less proline.

Methods for studying the amino acid composition

Proteins are broken down into amino acids by enzymes in the digestive juices. Two important conclusions were made: 1) proteins contain amino acids; 2) methods of hydrolysis can be used to study the chemical, in particular amino acid, composition of proteins.

To study the amino acid composition of proteins, a combination of acidic (HCl), alkaline [Ba(OH) 2 ], and, more rarely, enzymatic hydrolysis, or one of them, is used. It has been established 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 the qualitative and quantitative assessment of the amino acid composition of a given individual protein. It must be remembered that for the study you need to have a certain amount of pure protein, without impurities 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 cleaved 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 (for example, sulfonic acid residues -SO 3 -) strongly associated with it, to which Na + ions are attached (Fig. 1-4).

A mixture of amino acids is introduced into the cation exchanger in an acidic environment (pH 3.0), where the amino acids are mainly cations, i. carry a positive charge. Positively charged amino acids attach to negatively charged resin particles. The greater the total charge of the 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 bind the most weakly.

The release of amino acids from the column is carried out by eluting (eluting) them with a buffer solution with increasing ionic strength (ie, 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 exits the column at a specific pH and ionic strength. By collecting the solution (eluate) from the lower end of the column in the form of small portions, fractions containing individual amino acids can be obtained.

(for more details on "hydrolysis" see question #10)

8) Chemical bonds in the protein structure.

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

10) Protein hydrolysis. Reaction chemistry (stepping, catalysts, reagents, reaction conditions) - 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 the form of a random random coil to a greater or lesser extent. 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 acidic 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), and the biological activity of the protein is lost. Based on this, for example, the use of an aqueous solution of carbolic acid (phenol) as an antiseptic. Under certain conditions, with slow cooling of a solution of a denatured protein, renaturation occurs - the 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, such as heating or a significant change in pH, leads to a consistent violation 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, certain solvents (alcohol), radiation, etc.

Denaturation often leads to the process of aggregation of protein particles into larger ones in a colloidal solution of protein molecules. Visually, this looks, for example, as the formation of a "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 able to renature; in 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 one, then during renaturation, the ability of proteins to self-organize 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 decisive 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 reconnect into a regular double helix. This ability of DNA to renature is used to produce artificial DNA hybrid molecules.

Natural protein bodies are endowed with a certain, strictly defined spatial configuration and have a number of characteristic physicochemical and biological properties at physiological temperatures and pH values. Under the influence of various physical and chemical factors, proteins undergo coagulation and precipitate, losing their native properties. Thus, denaturation should be understood as a violation of the general plan 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 by the protein of its biological activity (catalytic, antigenic or hormonal). During protein denaturation caused by 8M urea or another agent, mostly non-covalent bonds (in particular, hydrophobic interactions and hydrogen bonds) are destroyed. Disulfide bonds are broken in the presence of the reducing agent mercaptoethanol, while the peptide bonds of the backbone 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 - beginning reversible violation of the molecular structure; c - irreversible deployment of the polypeptide chain.

Denaturation and renaturation of ribonuclease (according to Anfinsen).

a - deployment (urea + mercaptoethanol); b - refolding.

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. Precipitation of proteins:

a) reversible

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

b) irreversible (denaturation)

During denaturation under the action external factors(temperature; mechanical action - pressure, rubbing, shaking, ultrasound; the action of chemical agents - acids, alkalis, etc.) there is a change in the secondary, tertiary and quaternary structures of the protein macromolecule, i.e. its native spatial structure. Primary structure, and therefore chemical composition proteins do not change.

During denaturation, the physical properties of proteins change: solubility decreases, biological activity is lost. At the same time, the activity of some chemical groups increases, the effect of proteolytic enzymes on proteins is facilitated, and, consequently, it is more easily hydrolyzed.

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

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

3. Burning proteins

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

4. Color (qualitative) reactions to proteins:

a) xantoprotein reaction (for amino acid residues containing benzene rings):

Protein + HNO3 (conc.) → yellow color

b) biuret reaction (for peptide bonds):

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

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 various 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 dehydration of the protein particles and the removal of the charge occur, while the proteins precipitate. The degree of protein precipitation depends on the ionic strength of the precipitant solution, the size of the particles of the protein molecule, the magnitude of its charge, and hydrophilicity. Different proteins precipitate at different salt concentrations. Therefore, in sediments obtained by gradually increasing the concentration of salts, individual proteins are in different fractions. Salting out of proteins is a reversible process, and after the salt is removed, the protein regains its natural properties. Therefore, salting out is used in clinical practice in the separation of blood serum proteins, as well as in the isolation and purification of various proteins.

Added anions and cations destroy the hydrated protein shell of proteins, which is one of the stability factors of protein solutions. Most often, solutions of Na and ammonium sulfates are used. Many proteins differ in the size of the hydration shell and the magnitude of the charge. Each protein has its own salting out zone. After removal of the salting out agent, the protein retains its biological activity and physicochemical properties. In clinical practice, the salting out method is used to separate globulins (with the addition of 50% ammonium sulfate (NH4)2SO4 a precipitate precipitates) and albumins (with the addition of 100% ammonium sulfate (NH4)2SO4 a precipitate precipitates).

Salting out is influenced by:

1) nature and concentration of salt;

2) pH environments;

3) temperature.

The main role is played by the valencies of the ions.

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

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

Blood proteins regulate metabolic processes in the body, including those that occur inside cells. They also perform a transport function, since they carry nutrients, oxygen, and hormones to the cells. In addition, protein in the blood binds toxins, excess hormones, helps protect the body from pathogens, and performs other very important functions.

Proteins are called high-molecular organic substances circulating in plasma, the liquid part of the blood, which is 90% water, 6-8% protein, the rest is organic non-protein compounds and inorganic salts. Most proteins enter the body with food, after which they are broken down into amino acids. Some of them go to the creation of proteins, the rest are transformed into glucose or undergo other changes.

Most proteins are made up of twenty standard amino acids and non-amino acid groups in different combinations, which allows them to perform a huge number of tasks. For this reason, they are usually divided into two groups. Simple blood serum proteins consist only of polypeptide chains, while complex proteins also contain non-protein components, and therefore are divided into numerous types. Exactly how many of them are in the blood cannot be determined, as scientists are constantly discovering new compounds. The most famous of them are glycoproteins and lipoproteins.

Glycoproteins are proteins combined with carbohydrate residues of monosaccharides (glucose, fructose, etc.) and are important components of cell membranes. These include many protein hormones, receptor proteins, antibodies, and interferons, which make cells immune to the virus. Some glycoproteins on the membranes of red blood cells determine a person's blood type.

Lipoproteins contain lipids. Among them there are both water-soluble species (blood plasma lipoproteins) and insoluble ones (located on cell membranes, nerve fibers). The more soluble lipids in the composition, the lower the density of lipoproteins, and the worse they dissolve. One of the well-known functions of lipoproteins is the transport of water-insoluble cholesterol throughout the body.

Functions of proteins

Proteins perform a huge number of functions in the human body, so if the analysis showed their low or high content, this indicates violations. Proteins contribute to metabolic processes, muscle contraction, reduce the activity of other proteins, transmit signals and nutrients from cell to cell, take decay products and carry them to organs, which remove them from the body.

Enzyme proteins that accelerate certain reactions (there are more than 5 thousand species in the body). Structural proteins affect or change the shape of cells. For example, they include keratin, which makes up hair and nails, as well as collagen and elastin, the main components of the intercellular substance of connective tissue.

Some proteins protect a person: after detecting the presence of toxins in tissues, they bind them and bring them to the liver, which breaks down poisons, which allows them to be quickly removed from the body.

Thrombins and fibrinogens are involved in blood coagulation. Protection functions are performed by proteins of the complement system, as well as immunoglobulins (antibodies), which, after identifying pathogens or foreign cells, destroy them. Therefore, their low or high content in the blood indicates serious violations.

Transporter proteins help transport molecules through the cell's waterproof membrane. To do this, some proteins have fluid-filled pores through which water molecules pass through the membrane. Carrier proteins bind the necessary components and transport them into the cell using the energy of the ATP enzyme.

How to understand analysis data

A blood test for protein allows you to determine the presence of pathological disorders in the body. But other diagnostic tests will be needed to determine the cause. To determine the amount of proteins, clinical diagnostic centers use different methods of biochemical analysis. Among the methods of determination are the salting out method, as well as electrophoresis on paper.

The salting out method makes it possible to separate proteins into three fractions:

• Globulins characterize the state of immunity: antibodies, immunoglobulins, tumor necrosis factor and other protective proteins are produced from them.

• Fibrinogen - this fraction is responsible for the mechanism of human blood coagulation.

• Albumin - provide tissues in building material to maintain structure and produce new cells. It is present in the serum in the greatest amount.

Electrophoresis on paper allows you to make a separation into six fractions, additionally highlighting globulins. In this case, fibrinogen remains on paper.

In the body of women, the concentration of total protein is ten percent lower than that of male peers. The reason for this is that proteins are consumed much faster, since sex hormones are synthesized from them. In a pregnant woman, the indicators are even lower, and it is considered normal if the amount of protein is thirty percent below the permissible values. Also, the level of proteins is lower in the children's body, which is explained by rapid growth and development.

Depending on the reagents, the obtained values ​​​​of biochemical analysis in different clinical diagnostic centers may differ, therefore, first of all, you need to focus on the words of the doctor. Depending on the type of protein fraction, the following values ​​\u200b\u200bare considered normal in adults:

• Total protein: 64 to 84 g/l.
• Albumins: 35 to 55 g/l.
• Fibrinogen: 2 to 4 g/l.

Since the fraction of globulins includes several types, their amount is determined, if necessary, using electrophoresis on paper. If the decoding of biochemical analysis using paper electrophoresis or other separation method showed elevated levels of proteins in the blood, this may indicate disorders such as dehydration, increased production of antibodies due to vaccination or a recent illness. The reason for high values ​​may be a malignant tumor of plasma cells, as well as a violation of blood clotting due to too a large number platelets, which can be caused by poisoning and other critical situations.

The cause of low protein may be malnutrition or malnutrition caused by protein deficiency. Also, among the reasons, doctors call problems with the liver, kidney pathology. A value below the norm may indicate a chronic form of anemia, massive blood loss, diseases of the stomach, intestines, pancreas, thyroid gland. Also, decoding by electrophoresis can show the content of a low amount of protein in AIDS, oncology.

Thus, a protein deviation from the norm always signals a violation in the body. To determine the cause and make a diagnosis, in addition to a blood test for protein by salting out and electrophoresis, you need to undergo additional examinations at the clinical diagnostic center.

The necessary daily protein intake leads to the nutrition of muscle tissue and the correct level of amino acids in. Symptoms of an excess of protein in the body indicate tissue poisoning with its decay products, 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, so it is split into amino acids.

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

• Compounds are an integral part of the organelles and cytoplasms of body cells. For example, connective tissue protein is involved in the growth of hair, nail plates, and tendons.

Protein in the body plays an important role in the normal functioning of all organs. Therefore, it is very important to control the dose of protein. An excess of protein very often leads to serious diseases, therefore, at the first sign of a deviation, it is necessary to consult with specialists.