§ 9. PHYSICO-CHEMICAL PROPERTIES OF PROTEINS

Proteins are very large molecules, in size they can be inferior only to individual representatives of nucleic acids and polysaccharides. Table 4 presents the molecular characteristics of some proteins.

Table 4

Molecular characteristics of some proteins

	Relative molecular weight	Number of circuits	Number of amino acid residues
Ribonuclease
myoglobin
Chymotrypsin
Hemoglobin
Glutamate dehydrogenase

Protein molecules can contain a very different number of amino acid residues - from 50 to several thousand; the relative molecular masses of proteins also vary greatly - from several thousand (insulin, ribonuclease) to a million (glutamate dehydrogenase) or more. The number of polypeptide chains in proteins can range from one to several tens or even thousands. Thus, the tobacco mosaic virus protein contains 2120 protomers.

Knowing the relative molecular weight of a protein, one can approximately estimate how many amino acid residues are included in its composition. The average relative molecular weight of the amino acids that form the polypeptide chain is 128. When a peptide bond is formed, a water molecule is cleaved off, therefore, the average relative mass of the amino acid residue will be 128 - 18 = 110. Using these data, we can calculate that a protein with a relative molecular weight of 100,000 will consist of approximately 909 amino acid residues.

Electrical properties of protein molecules

The electrical properties of proteins are determined by the presence of positively and negatively charged amino acid residues on their surface. The presence of charged protein groups determines the total charge of the protein molecule. If negatively charged amino acids predominate in proteins, then its molecule in a neutral solution will have a negative charge, if positively charged amino acids predominate, the molecule will have a positive charge. The total charge of the protein molecule also depends on the acidity (pH) of the medium. With an increase in the concentration of hydrogen ions (an increase in acidity), the dissociation of carboxyl groups is suppressed:

and at the same time, the number of protonated amino groups increases;

Thus, with an increase in the acidity of the medium, the number of negatively charged groups on the surface of the protein molecule decreases and the number of positively charged groups increases. A completely different picture is observed with a decrease in the concentration of hydrogen ions and an increase in the concentration of hydroxide ions. The number of dissociated carboxyl groups increases

and the number of protonated amino groups decreases

So, by changing the acidity of the medium, the charge of the protein molecule can also be changed. With an increase in the acidity of the medium in the protein molecule, the number of negatively charged groups decreases and the number of positively charged groups increases, the molecule gradually loses the negative and acquires a positive charge. With a decrease in the acidity of the solution, the opposite picture is observed. Obviously, at certain pH values, the molecule will be electrically neutral; the number of positively charged groups will be equal to the number of negatively charged groups, and the total charge of the molecule will be zero (Fig. 14).

The pH value at which the total charge of the protein is zero is called the isoelectric point and is denotedpi.

Rice. 14. In the state of the isoelectric point, the total charge of the protein molecule is zero

The isoelectric point for most proteins is in the pH range of 4.5 to 6.5. However, there are exceptions. Below are the isoelectric points of some proteins:

At pH values ​​below the isoelectric point, the protein carries a total positive charge, and above it, a total negative charge.

At the isoelectric point, the solubility of the protein is minimal, since its molecules in this state are electrically neutral and there are no mutual repulsion forces between them, so they can “stick together” due to hydrogen and ionic bonds, hydrophobic interactions, van der Waals forces. At pH values ​​different from pI, protein molecules will carry the same charge - either positive or negative. As a result of this, electrostatic repulsion forces will exist between the molecules, preventing them from “sticking together”, the solubility will be higher.

Protein solubility

Proteins are soluble and insoluble in water. The solubility of proteins depends on their structure, pH value, salt composition of the solution, temperature and other factors and is determined by the nature of those groups that are on the surface of the protein molecule. Insoluble proteins include keratin (hair, nails, feathers), collagen (tendons), fibroin (lye, cobweb). Many other proteins are water soluble. Solubility is determined by the presence of charged and polar groups on their surface (-COO -, -NH 3 +, -OH, etc.). Charged and polar groupings of proteins attract water molecules to themselves, and a hydration shell is formed around them (Fig. 15), the existence of which determines their solubility in water.

Rice. 15. Formation of a hydration shell around a protein molecule.

Protein solubility is affected by the presence of neutral salts (Na 2 SO 4 , (NH 4) 2 SO 4 , etc.) in solution. At low salt concentrations, protein solubility increases (Fig. 16), since under such conditions the degree of dissociation of polar groups increases and the charged groups of protein molecules are shielded, thereby reducing the protein-protein interaction, which contributes to the formation of aggregates and protein precipitation. At high salt concentrations, protein solubility decreases (Fig. 16) due to the destruction of the hydration shell, leading to aggregation of protein molecules.

Rice. 16. Dependence of protein solubility on salt concentration

There are proteins that dissolve only in salt solutions and do not dissolve in pure water, such proteins are called globulins. There are other proteins albumins, unlike globulins, they are highly soluble in pure water.
The solubility of proteins also depends on the pH of the solutions. As we have already noted, proteins have minimal solubility at the isoelectric point, which is explained by the absence of electrostatic repulsion between protein molecules.
Under certain conditions, proteins can form gels. During the formation of a gel, protein molecules form a dense network, the interior of which is filled with a solvent. Gels form, for example, gelatin (this protein is used to make jelly) and milk proteins in the preparation of curdled milk.
The temperature also affects the solubility of the protein. Under the action of high temperature, many proteins precipitate due to the disruption of their structure, but this will be discussed in more detail in the next section.

Protein denaturation

Let us consider a well-known phenomenon. When the egg white is heated, it gradually becomes cloudy, and then a solid clot forms. Coagulated egg white - egg albumin - after cooling is insoluble, while before heating the egg white is highly soluble in water. The same phenomena occur when almost all globular proteins are heated. The changes that occur during heating are called denaturation. Proteins in their natural state are called native proteins, and after denaturation - denatured.
During denaturation, the native conformation of proteins is violated as a result of breaking weak bonds (ionic, hydrogen, hydrophobic interactions). As a result of this process, the quaternary, tertiary and secondary structures of the protein can be destroyed. The primary structure is preserved (Fig. 17).

Rice. 17. Protein denaturation

During denaturation, hydrophobic amino acid radicals, which are found in native proteins in the depth of the molecule, appear on the surface, as a result, conditions for aggregation are created. Aggregates of protein molecules precipitate. Denaturation is accompanied by the loss of the biological function of the protein.

Protein denaturation can be caused not only by elevated temperature, but also by other factors. Acids and alkalis can cause protein denaturation: as a result of their action, the ionogenic groups are recharged, which leads to the breaking of ionic and hydrogen bonds. Urea destroys hydrogen bonds, which results in the loss of their native structure by proteins. Denaturing agents are organic solvents and heavy metal ions: organic solvents destroy hydrophobic bonds, and heavy metal ions form insoluble complexes with proteins.

Along with denaturation, there is also a reverse process - renaturation. With the removal of the denaturing factor, it is possible to restore the original native structure. For example, when the solution is slowly cooled to room temperature, the native structure and biological function of trypsin are restored.

Proteins can also be denatured in the cell during normal life processes. It is quite obvious that the loss of the native structure and function of proteins is an extremely undesirable event. In this regard, special proteins should be mentioned - chaperones. These proteins are able to recognize partially denatured proteins and, by binding to them, restore their native conformation. Chaperones also recognize proteins that are far from denaturing and transport them to lysosomes where they are degraded. Chaperones also play an important role in the formation of tertiary and quaternary structures during protein synthesis.

Interesting to know! Currently, such a disease as mad cow disease is often mentioned. This disease is caused by prions. They can also cause other neurodegenerative diseases in animals and humans. Prions are proteinaceous infectious agents. When a prion enters a cell, it causes a change in the conformation of its cellular counterpart, which itself becomes a prion. This is how disease occurs. The prion protein differs from the cellular protein in its secondary structure. The prion form of the protein is mainlyb-folded structure, and cellular -a- spiral.

Donetsk secondary school I - III levels No. 21

"Squirrels. Obtaining proteins by the reaction of polycondensation of amino acids. Primary, secondary and tertiary structures of proteins. Chemical properties of proteins: combustion, denaturation, hydrolysis and color reactions. Biochemical functions of proteins”.

Prepared

chemistry teacher

teacher - methodologist

Donetsk, 2016

“Life is a way of existence of protein bodies”

Lesson topic. Squirrels. Obtaining proteins by the reaction of polycondensation of amino acids. Primary, secondary and tertiary structures of proteins. Chemical properties of proteins: combustion, denaturation, hydrolysis and color reactions. Biochemical functions of proteins.

Lesson goals. To acquaint students with proteins as the highest degree of development of substances in nature that led to the emergence of life; show their structure, properties and variety of biological functions; to expand the concept of the polycondensation reaction using the example of obtaining proteins, to inform schoolchildren about food hygiene, about maintaining their health. Develop logical thinking in students.

Reagents and equipment. Table "Primary, secondary and tertiary structures of proteins". Reagents: HNO3, NaOH, CuSO4, chicken protein, woolen thread, chemical glassware.

lesson method. Information and development.

Lesson type. A lesson in mastering new knowledge and skills.

During the classes

I. Organizing time.

II. Checking homework, updating and correcting basic knowledge.

Blitz poll

1. Explain the term "amino acid".

2. Name the functional groups that make up the amino acids.

3. Nomenclature of amino acids and their isomerism.

4. Why do amino acids exhibit amphoteric properties? Write the equations of chemical reactions.

5. Due to what properties amino acids form polypeptides. Write the reaction for the polycondensation of amino acids.

III. The message of the topic, the objectives of the lesson, the motivation of educational activities.

IV. Perception and initial awareness of new material.

Teacher.

“Wherever we meet life, we find that it is associated with some kind of protein body,” wrote F. Engels in his book “Anti-Dühring”. Lack of protein in food leads to a general weakening of the body, in children - to a slowdown in mental and physical development. Today, more than half of humanity does not receive the required amount of protein from food. A person needs 115 g of protein per day, protein is not stored in reserve, unlike carbohydrates and fats, so you need to monitor your diet. We are familiar with keratin - the protein that makes up hair, nails, feathers, skin - it performs a building function; familiar with the protein pepsin - it is found in gastric juice and is able to destroy other proteins during digestion; the thrombin protein is involved in blood clotting; pancreatic hormone - insulin - regulates glucose metabolism; hemoglobin transports O2 to all cells and tissues of the body, etc.

Where does this endless variety of protein molecules come from, the variety of their functions and their special role in life processes? In order to answer this question, let us turn to the composition and structure of proteins.

Are proteins made up of atoms?

To answer this question, let's do a warm-up. Guess the riddles and explain the meaning of the answers.

1. He is everywhere and everywhere:

In stone, in air, in water.

He is in the morning dew

And blue in the sky.

(oxygen)

2. I am the lightest element,

In nature, not a step without me.

And with oxygen I'm at the moment

3. In the air, it is the main gas,

Surrounds us everywhere.

Plant life is fading

Without it, without fertilizer.

Lives in our cells

4. Schoolchildren went on a hike

(This is the approach to the chemical problem).

At night, a fire was lit by the moon,

Songs were sung about bright fire.

Throw aside your sentiments:

What elements burned in the fire?

(carbon, hydrogen)

Yes, that's right, these are the main chemical elements that make up the protein.

These four elements can be said in Schiller's words, "Four elements, merging together, give life and build the world."

Proteins are natural polymers consisting of α-amino acid residues linked by peptide bonds.

The composition of proteins includes 20 different amino acids, hence the huge variety of proteins in their various combinations. There are up to 100,000 proteins in the human body.

History reference.

The first hypothesis about the structure of the protein molecule was proposed in the 70s. 19th century This was the ureide theory of protein structure.

In 1903 German scientists expressed the peptide theory, which gave the key to the mystery of the structure of the protein. Fisher suggested that proteins are polymers of amino acids linked by peptide bonds.

The idea that proteins are polymeric formations was expressed as early as 70-88 years. 19th century , Russian scientists. This theory has been confirmed in modern works.

Even the first acquaintance with proteins gives some idea of ​​the extremely complex structure of their molecules. Proteins are obtained by the polycondensation reaction of amino acids:

https://pandia.ru/text/80/390/images/image007_47.gif" width="16" height="18">H - N - CH2 - C + H - N - CH2 - C →

https://pandia.ru/text/80/390/images/image012_41.gif" height="20">

NH2 - CH - C - N - CH - C - N - CH - C - ... + nH2O →

⸗ O ⸗ O ⸗ O

→ NH2 – CH – C + NH2 – CH – C + NH2 – CH – C + …

̀ OH ̀ OH ̀ OH

4. The teacher demonstrates the experience: burning a woolen thread; there is a smell of scorched feathers - this is how you can distinguish wool from fabrics of other types.

V. Generalization and systematization of knowledge.

1. Make a basic summary of proteins.

basis of life ← Proteins → polypeptides

(C, H, O, N) ↓ ↓ ↓ \ protein structures

chemical color functions

which properties of protein reactions

2. Write the reaction equations for the formation of a dipeptide from glycine and valine.

VI. Summing up the lesson, homework.

Learn §38 p. 178 - 184. Complete test tasks p. 183.

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 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 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 in an 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 of the protein. 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. The chemical and physical properties of proteins are due to the chemical nature and physico-chemical properties of the radicals that make up their 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 the physical and chemical properties of these compounds.

Proteins when interacting with certain chemicals give colored compounds. The formation of these compounds occurs with the participation of amino acid radicals, their specific groups or peptide bonds. Color reactions 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 solutions of acids (0.1 N), are distinguished by a high content of amino acids: arginine, lysine and histidine (at least 30%) and therefore have basic properties. These proteins are found in significant 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 acid groups of amino acid radicals, replacing their own intramolecular hydrogen bonds in the protein, as a result of which the secondary and tertiary structures change. During denaturation, the solubility of the protein decreases, it “coagulates” (for example, when boiling a chicken egg), 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 state, 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 - 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, mainly 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 influence of external factors (temperature; mechanical action - pressure, rubbing, shaking, ultrasound; the action of chemical agents - acids, alkalis, etc.), a change occurs in the secondary, tertiary and quaternary structures of the protein macromolecule, i.e. its native spatial structure. The primary structure, and, consequently, the chemical composition of the protein does 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 forms) and albumins (with the addition of 100% ammonium sulfate (NH4)2SO4 a precipitate forms).

Salting out is influenced by:

1) nature and concentration of salt;

2) pH environments;

3) temperature.

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

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.

The amino acid composition and spatial organization of each protein determine its physicochemical properties. Proteins have acid-base, buffer, colloidal and osmotic properties.

Proteins as amphoteric macromolecules

Proteins are amphoteric polyelectrolytes, i.e. combine, like amino acids, acidic and basic properties. However, the nature of the groups that give amphoteric properties to proteins is far from being the same as that of amino acids. The acid-base properties of amino acids are primarily due to the presence of α-amino and α-carboxyl groups (acid-base pair). In protein molecules, these groups participate in the formation of peptide bonds, and amphoteric proteins are given by acid-base groups of side radicals of amino acids that make up the protein. Of course, in each native protein molecule (polypeptide chain) there is at least one terminal α-amino and α-carboxyl group (if the protein has only a tertiary structure). In a protein with a quaternary structure, the number of end groups -NH 2 and -COOH is equal to the number of subunits, or protomers. However, such a small number of these groups cannot explain the amphoteric nature of protein macromolecules. Since most of the polar groups are located on the surface of globular proteins, they determine the acid-base properties and charge of the protein molecule. Acidic properties of the protein are given by acidic amino acids (aspartic, glutamic and aminocitric), and alkaline properties are given by basic amino acids (lysine, arginine, histidine). The more acidic amino acids a protein contains, the more pronounced its acid properties are, and the more basic amino acids are included in the protein, the stronger its basic properties are manifested. Weak dissociation of the SH group of cysteine ​​and the phenolic group of tyrosine (they can be considered as weak acids) has almost no effect on the amphotericity of proteins.

Buffer Properties. 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 side group of histidine has buffer properties in the pH range close to physiological. There are very few of these proteins. Hemoglobin is almost the only protein containing up to 8% histidine, which is a powerful intracellular buffer in erythrocytes, maintaining the blood pH at a constant level.

The charge of a protein molecule depends on the content of acidic and basic amino acids in it, or rather, on the ionization of the acidic and basic groups of the side radical of these amino acids. The dissociation of the COOH groups of acidic amino acids causes a negative charge to appear on the surface of the protein, and the side radicals of alkaline amino acids carry a positive charge (due to the addition of H + to the main groups). In a native protein molecule, charges are distributed asymmetrically depending on the spatial arrangement of the polypeptide chain. If acidic amino acids predominate over basic ones in a protein, then in general the protein molecule is electronegative, that is, it is a polyanion, and vice versa, if basic amino acids predominate, then it is positively charged, i.e., it behaves like a polycation.

The total charge of a protein molecule, of course, depends on the pH of the medium: in an acidic medium it is positive, in an alkaline medium it is negative. The pH value at which the protein has a net charge of zero is called the isoelectric point of the protein. At this point, the protein does not have mobility in the electric field. The isoelectric point of each protein is determined by the ratio of acidic and basic groups of amino acid side radicals: the higher the ratio of acidic/basic amino acids in a protein, the lower its isoelectric point. Acidic proteins have a pH of 1< 7, у нейтральных рН 1 около 7, а у основных рН 1 >7. At pH values ​​below its isoelectric point, the protein will carry a positive charge, and above - a negative charge. The average isoelectric point of all cytoplasmic proteins lies within 5.5. Therefore, at physiological pH (about 7.0 - 7.4), cellular proteins have an overall negative charge. The excess of negative charges of proteins inside the cell is balanced, as already mentioned, by inorganic cations.

Knowing the isoelectric point is very important for understanding the stability of proteins in solutions, since proteins are the least stable in the isoelectric state. Uncharged protein particles can stick together and precipitate.

Colloidal and osmotic properties of proteins

The behavior of proteins in solutions has some peculiarities. Ordinary colloidal solutions are stable only in the presence of a stabilizer that prevents the colloids from settling down at the solute-solvent interface.

Aqueous solutions of proteins are stable and balanced; they do not precipitate (do not coagulate) over time and do not require the presence of stabilizers. Protein solutions are homogeneous and, in essence, they can be classified as true solutions. However, the high molecular weight of proteins gives their solutions many properties of colloidal systems:

• characteristic optical properties (opalescence of solutions and their ability to scatter visible light rays) [show] .

  Optical properties of proteins. Solutions of proteins, especially concentrated ones, have a characteristic opalescence. When the protein solution is illuminated sideways, the light rays in it become visible and form a luminous cone or strip - the Tyndall effect (in highly diluted protein solutions, opalescence is not visible and the luminous Tyndall cone is almost absent). This light-scattering effect is explained by the diffraction of light rays by protein particles in solution. It is believed that in the protoplasm of the cell, the protein is in the form of a colloidal solution - a sol. The ability of proteins and other biological molecules (nucleic acids, polysaccharides, etc.) to scatter light is used in the microscopic study of cellular structures: in the dark field of a microscope, colloidal particles are visible as light blotches in the cytoplasm.

  The light-scattering ability of proteins and other macromolecular substances is used for their quantitative determination by nephelometry, comparing the intensity of light scattering by suspended particles of the test and standard sol.

• low diffusion rate [show] .

  Low diffusion rate. Diffusion is the spontaneous movement of solute molecules due to a concentration gradient (from areas of high concentration to areas of low concentration). Proteins have a limited diffusion rate compared to ordinary molecules and ions, which move hundreds to thousands of times faster than proteins. The diffusion rate of proteins depends more on the shape of their molecules than on their molecular weight. Globular proteins in aqueous solutions are more mobile than fibrillar proteins.

  The diffusion of proteins is essential for the normal functioning of the cell. The synthesis of proteins in any part of the cell (where there are ribosomes) could lead, in the absence of diffusion, to the accumulation of proteins at the site of their formation. Intracellular distribution of proteins occurs by diffusion. Since the rate of protein diffusion is low, it limits the rate of processes that depend on the function of the diffusing protein in the corresponding area of ​​the cell.

• inability to penetrate semipermeable membranes [show] .

  Osmotic properties of proteins. Proteins, due to their high molecular weight, cannot diffuse through a semipermeable membrane, while low molecular weight substances easily pass through such membranes. This property of proteins is used in practice to purify their solutions from low-molecular impurities. This process is called dialysis.

  The inability of proteins to diffuse through semipermeable membranes causes the phenomenon of osmosis, i.e., the movement of water molecules through a semipermeable membrane into a protein solution. If the protein solution is separated from the water by a cellophane membrane, then, striving to achieve equilibrium, water molecules diffuse into the protein solution. However, the movement of water into the space where the protein is located increases the hydrostatic pressure in it (the pressure of the water column), which prevents further diffusion of water molecules to the protein.

  The pressure or force that must be applied to stop the osmotic flow of water is called osmotic pressure. The osmotic pressure in very dilute protein solutions is proportional to the molar concentration of the protein and to the absolute temperature.

  Biological membranes are also impermeable to protein, so the osmotic pressure created by the protein depends on its concentration inside and outside the cell. The osmotic pressure due to protein is also called oncotic pressure.

• high viscosity solutions [show] .

  High viscosity protein solutions. High viscosity is typical not only for protein solutions, but in general for solutions of macromolecular compounds. With an increase in protein concentration, the viscosity of the solution increases, since the adhesion forces between protein molecules increase. Viscosity depends on the shape of the molecules. Solutions of fibrillar proteins are always more viscous than solutions of globular proteins. The viscosity of solutions is strongly affected by temperature and the presence of electrolytes. As the temperature increases, the viscosity of protein solutions decreases. Additions of some salts, such as calcium, increase the viscosity by promoting the adhesion of molecules with the help of calcium bridges. Sometimes the viscosity of the protein solution increases so much that it loses fluidity and passes into a gel-like state.

• gelling ability [show] .

  The ability of proteins to form gels. The interaction between protein macromolecules in solution can lead to the formation of structural networks, inside which are trapped water molecules. Such structured systems are called gels or jellies. It is believed that the protein of the protoplasm of the cell can pass into a gel-like state. A typical example - the body of a jellyfish is like a living jelly, the water content of which is up to 90%.

  Gelation proceeds more easily in solutions of fibrillar proteins; their rod-shaped form promotes better contact of the ends of macromolecules. This is well known from everyday practice. Food jellies are prepared from products (bones, cartilage, meat) containing large amounts of fibrillar proteins.

  In the process of life of the body, the gel-like state of protein structures is of great physiological importance. Collagen proteins of bones, tendons, cartilage, skin, etc. have high strength, firmness and elasticity, because they are in a gel-like state. The deposition of mineral salts during aging reduces their firmness and elasticity. In a gel-like or gelatinous form, actomyosin is found in muscle cells, which performs a contractile function.

  In a living cell, processes resembling a sol-gel transition occur. The protoplasm of a cell is a sol-like viscous liquid, in which islands of gel-like structures are found.

Hydration of proteins and factors affecting their solubility

Proteins are hydrophilic substances. If you dissolve a dry protein in water, then at first it, like any hydrophilic high-molecular compound, swells, and then the protein molecules begin to gradually pass into the solution. During swelling, water molecules penetrate the protein and bind to its polar groups. The dense packing of polypeptide chains is loosened. A swollen protein can be considered as a back solution, i.e., a solution of water molecules in a high molecular weight substance - protein. Further absorption of water leads to the separation of protein molecules from the total mass and dissolution. But swelling does not always lead to dissolution; some proteins, such as collagen, remain swollen after absorbing large amounts of water.

Dissolution is associated with the hydration of proteins, i.e., the binding of water molecules to proteins. Hydrated water is so strongly bound to the protein macromolecule that it is difficult to separate it. This indicates not simple adsorption, but electrostatic binding of water molecules with polar groups of side radicals of acidic amino acids bearing a negative charge and basic amino acids bearing a positive charge.

However, part of the water of hydration is bound by peptide groups, which form hydrogen bonds with water molecules. For example, polypeptides with non-polar side groups also swell, i.e., bind water. Thus, a large amount of water binds collagen, although this protein contains predominantly non-polar amino acids. Water, by binding to the peptide groups, pushes the elongated polypeptide chains apart. However, interchain bonds (bridges) do not allow protein molecules to break away from each other and go into solution. When raw materials containing collagen are heated, interchain bridges in collagen fibers break and the released polypeptide chains pass into solution. This fraction of partially hydrolyzed soluble collagen is called gelatin. Gelatin is similar in chemical composition to collagen, easily swells and dissolves in water, forming viscous liquids. A characteristic property of gelatin is the ability to gel. Aqueous solutions of gelatin are widely used in medical practice as a plasma-substituting and hemostatic agent, and the ability to gel - in the manufacture of capsules in pharmaceutical practice.

Factors affecting the solubility of proteins. The solubility of different proteins varies widely. It is determined by their amino acid composition (polar amino acids give greater solubility than non-polar ones), organizational features (globular proteins are usually better soluble than fibrillar ones), and solvent properties. For example, vegetable proteins - prolamins - dissolve in 60-80% alcohol, albumins - in water and in weak salt solutions, and collagen and keratins are insoluble in most solvents.

Protein solutions are stable due to the charge of the protein molecule and the hydration shell. Each macromolecule of an individual protein has a total charge of the same sign, which prevents them from sticking together in solution and precipitating. Anything that contributes to the conservation of charge and hydration shell facilitates the solubility of the protein and its stability in solution. There is a close relationship between the charge of a protein (or the number of polar amino acids in it) and hydration: the more polar amino acids in a protein, the more water binds (per 1 g of protein). The hydration shell of a protein sometimes reaches a large size, and water of hydration can be up to 1/5 of its mass.

True, some proteins are more hydrated and less soluble. For example, collagen binds water more than many highly soluble globular proteins, but does not dissolve. Its solubility is hindered by structural features - cross-links between polypeptide chains. Sometimes oppositely charged protein groups form many ionic (salt) bonds within the protein molecule or between protein molecules, which prevents the formation of bonds between water molecules and charged protein groups. A paradoxical phenomenon is observed: there are many anionic or cationic groups in the protein, and its solubility in water is low. Intermolecular salt bridges cause protein molecules to stick together and precipitate.

What environmental factors affect the solubility of proteins and their stability in solutions?

• Influence of neutral salts [show] .

  Neutral salts in small concentrations increase the solubility of even those proteins that are insoluble in pure water (for example, euglobulins). This is due to the fact that salt ions, interacting with oppositely charged groups of protein molecules, destroy salt bridges between protein molecules. Increasing the concentration of salts (increasing the ionic strength of the solution) has the opposite effect (see below - salting out).

• Influence of medium pH [show] .

  The pH of the medium affects the charge of the protein and, consequently, its solubility. The least stable protein is in the isoelectric state, i.e., when its total charge is zero. Removing the charge allows protein molecules to easily approach each other, stick together and precipitate. This means that the solubility and stability of the protein will be minimal at pH corresponding to the isoelectric point of the protein.

• Temperature effect [show] .

  There is no strict relationship between temperature and the nature of protein solubility. Some proteins (globulins, pepsin, muscle phosphorylase) in aqueous or saline solutions dissolve better with increasing temperature; others (muscle aldolase, hemoglobin, etc.) are worse.

• Influence of differently charged protein [show] .

  If a protein that is a polycation (basic protein) is added to a solution of a protein that is a polyanion (acidic protein), then they form aggregates. In this case, the stability due to the neutralization of charges is lost and the proteins precipitate. Sometimes this feature is used to isolate the desired protein from a mixture of proteins.

salting out

Solutions of neutral salts are widely used not only to increase the solubility of a protein, for example, when isolating it from biological material, but also for the selective precipitation of various proteins, i.e., their fractionation. The process of precipitation of proteins by neutral saline solutions is called salting out. A characteristic feature of proteins obtained by salting out is that they retain their native biological properties after salt removal.

The mechanism of salting out is that the added anions and cations of the saline solution remove the hydration shell of proteins, which is one of the factors of its stability. Possibly, the neutralization of protein charges by salt ions occurs simultaneously, which also contributes to the precipitation of proteins.

The ability to salt out is most pronounced in salt anions. According to the strength of the salting-out action, anions and cations are arranged in the following rows:

• SO 4 2-> C 6 H 5 O 7 3-> CH 3 COO - > Cl - > NO 3 - > Br - > I - > CNS -
• Li + >Na + > K + > Pb + > Cs +

These series are called lyotropic.

Sulfates have a strong salting-out effect in this series. In practice, sodium and ammonium sulfate are most often used for salting out proteins. In addition to salts, proteins are precipitated with organic water-removing agents (ethanol, acetone, methanol, etc.). In fact, this is the same salting out.

Salting out is widely used to separate and purify proteins, since many proteins differ in the size of their hydration shell and the magnitude of their charges. Each of them has its own salting out zone, i.e., the salt concentration that allows dehydration and precipitation of the protein. After removal of the salting out agent, the protein retains all its natural properties and functions.

Denaturation (denativation) and renaturation (renativation)

Under the action of various substances that violate the highest levels of organization of the protein molecule (secondary, tertiary, quaternary) while maintaining the primary structure, the protein loses its native physicochemical and, most importantly, biological properties. This phenomenon is called denaturation (denativation). It is characteristic only for molecules having a complex spatial organization. Synthetic and natural peptides are not capable of denaturation.

During denaturation, the bonds that stabilize the quaternary, tertiary, and even secondary structures are broken. The polypeptide chain unfolds and is in solution either in an unfolded form or in the form of a random coil. In this case, the hydration shell is lost and the protein precipitates. However, the precipitated denatured protein differs from the same protein precipitated by salting out, since in the first case it loses its native properties, while in the second it retains. This indicates that the mechanism of action of substances that cause denaturation and salting out is different. During salting out, the native structure of the protein is preserved, and during denaturation it is destroyed.

Denaturing factors are divided into

• physical [show] .

  Physical factors include: temperature, pressure, mechanical impact, ultrasonic and ionizing radiation.

  Thermal denaturation of proteins is the most studied process. It was considered one of the characteristic features of proteins. It has long been known that when heated, the protein coagulates (coagulates) and precipitates. Most proteins are thermolabile, but proteins are known to be very resistant to heat. For example, trypsin, chymotrypsin, lysozyme, some biological membrane proteins. Proteins of bacteria living in hot springs are especially resistant to temperature. Obviously, in thermostable proteins, the thermal motion of polypeptide chains caused by heating is not sufficient to break the internal bonds of protein molecules. At the isoelectric point, proteins are more easily denatured by heat. This technique is used in practical work. Some proteins, on the other hand, denature at low temperatures.

• chemical [show] .

  Chemical factors that cause denaturation include: acids and alkalis, organic solvents (alcohol, acetone), detergents (detergents), some amides (urea, guanidine salts, etc.), alkaloids, heavy metals (salts of mercury, copper , barium, zinc, cadmium, etc.). The mechanism of the denaturing action of chemicals depends on their physicochemical properties.

  Acids and alkalis are widely used as protein precipitants. Many proteins denature at extreme pH values ​​below 2 or above 10-11. But some proteins are resistant to acids and alkalis. For example, histones and protamines do not denature even at pH 2 or pH 10. Strong solutions of ethanol and acetone also have a denaturing effect on proteins, although for some proteins these organic solvents are used as salting out agents.

  Heavy metals, alkaloids have long been used as precipitants; they form strong bonds with the polar groups of proteins and thereby break the system of hydrogen and ionic bonds.

  Particular attention should be paid to urea and guanidine salts, which in high concentrations (for urea 8 mol/l, for guanidine hydrochloride 2 mol/l) compete with peptide groups for the formation of hydrogen bonds. As a result, dissociation into subunits occurs in proteins with a quaternary structure, and then the unfolding of polypeptide chains. This property of urea is so striking that it is widely used to prove the presence of a quaternary protein structure and the importance of its structural organization in the implementation of a physiological function.

Properties of denatured proteins . The most typical for denatured proteins are the following features.

• An increase in the number of reactive or functional groups compared to the native protein molecule (functional groups are groups of side radicals of amino acids: COOH, NH 2, SH, OH). Some of these groups are usually located inside the protein molecule and are not detected by special reagents. The unfolding of the polypeptide chain during denaturation reveals these additional, or hidden, groups.
• Decreased solubility and precipitation of the protein (associated with the loss of the hydration shell, the unfolding of the protein molecule with the "exposure" of hydrophobic radicals and the neutralization of the charges of polar groups).
• Change in the configuration of a protein molecule.
• Loss of biological activity caused by a violation of the native structural organization of the molecule.
• Easier cleavage by proteolytic enzymes compared to native protein, the transition of a compact native structure into an unfolded loose form facilitates the access of enzymes to the peptide bonds of the protein, which they destroy.

The latter quality of denatured protein is widely known. Thermal or other processing of products containing proteins (mainly meat) contributes to their better digestion with the help of proteolytic enzymes of the gastrointestinal tract. In the stomach of humans and animals, a natural denaturing agent is produced - hydrochloric acid, which, by denaturing proteins, helps them to be broken down by enzymes. However, the presence of hydrochloric acid and proteolytic enzymes does not allow the use of protein drugs through the mouth, because they are denatured and immediately split, losing their biological activity.

We also note that denaturing substances that precipitate proteins are used in biochemical practice for other purposes than salting out ones. Salting out as a technique is used to isolate a certain protein or group of proteins, and denaturation is used to free a mixture of any substances from a protein. By removing the protein, a protein-free solution can be obtained or the effect of this protein can be eliminated.

It has long been believed that denaturation is irreversible. However, in some cases, the removal of the denaturing agent (such experiments were made using urea) restores the biological activity of the protein. The process of restoring the physicochemical and biological properties of a denatured protein is called renaturation or renativation. If the denatured protein (after the removal of denaturing substances) re-organizes itself into the original structure, then its biological activity is restored.

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The shape of the protein molecule. Studies of the native conformation of protein molecules have shown that these particles in most cases have a more or less asymmetric shape. Depending on the degree of asymmetry, i.e., the ratio between the long (b) and short (a) axes of the protein molecule, globular (spherical) and fibrillar (filamentous) proteins are distinguished.

Globular are protein molecules in which the folding of polypeptide chains has led to the formation of a spherical structure. Among them there are strictly spherical, elliptical and rod-shaped. They differ in the degree of asymmetry. For example, egg albumin has b/a = 3, wheat gliadin has 11, and corn zein has 20. Many proteins in nature are globular.

Fibrillar proteins form long, highly asymmetric filaments. Many of them have a structural or mechanical function. These are collagen (b / a - 200), keratins, fibroin.

Proteins of each group have their own characteristic properties. Many globular proteins are soluble in water and dilute saline solutions. Soluble fibrillar proteins are characterized by very viscous solutions. Globular proteins, as a rule, have a good biological value - they are absorbed during digestion, while many fibrillar proteins are not.

There is no clear boundary between globular and fibrillar proteins. A number of proteins occupy an intermediate position and combine features of both globular and fibrillar ones. Such proteins include, for example, muscle myosin (b/a = 75) and blood fibrinogen (b/a = 18). Myosin has a rod-like shape, similar to the shape of fibrillar proteins, however, like globular proteins, it is soluble in saline solutions. Solutions of myosin and fibrinogen are viscous. These proteins are absorbed during digestion. At the same time, actin, a globular muscle protein, is not absorbed.

Protein denaturation. The native conformation of protein molecules is not rigid, it is rather labile (lat. "labilis" - sliding) and can be seriously disturbed under a number of influences. Violation of the native conformation of a protein, accompanied by a change in its native properties without breaking peptide bonds, is called denaturation (Latin "denaturare" - to deprive the natural properties) of the protein.

Protein denaturation can be caused by various reasons leading to the disruption of weak interactions, as well as to the breaking of disulfide bonds that stabilize their native structure.

Heating most proteins to temperatures above 50°C, as well as ultraviolet and other types of high-energy irradiation, increase the vibrations of the atoms of the polypeptide chain, which leads to the disruption of various bonds in them. Even mechanical shaking can cause protein denaturation.

Protein denaturation also occurs due to chemical attack. Strong acids or alkalis affect the ionization of acidic and basic groups, causing the disruption of ionic and some hydrogen bonds in protein molecules. Urea (H 2 N-CO-NH 2) and organic solvents - alcohols, phenols, etc. - break the system of hydrogen bonds and weaken hydrophobic interactions in protein molecules (urea - due to a violation of the structure of water, organic solvents - due to the establishment of contacts with non-polar amino acid radicals). Mercaptoethanol destroys disulfide bonds in proteins. Heavy metal ions disrupt weak interactions.

During denaturation, a change in the properties of the protein occurs and, first of all, a decrease in its solubility. For example, when boiled, proteins coagulate and precipitate from solutions in the form of clots (as when boiling a chicken egg). Precipitation of proteins from solutions also occurs under the influence of protein precipitants, which are used as trichloroacetic acid, Barnstein's reagent (a mixture of sodium hydroxide with copper sulfate), tannin solution, etc.

During denaturation, the water-absorbing capacity of the protein decreases, i.e., its ability to swell; new chemical groups may appear, for example: when exposed to measures of captoethanol - SH-groups. As a result of denaturation, the protein loses its biological activity.

Although the primary structure of a protein is not affected by denaturation, the changes are irreversible. However, for example, with the gradual removal of urea by dialysis from a solution of denatured protein, its renaturation occurs: the native structure of the protein is restored, and along with it, to one degree or another, its native properties. Such denaturation is called reversible.

Irreversible denaturation of proteins occurs during the aging of organisms. Therefore, for example, plant seeds, even under optimal storage conditions, gradually lose their germination capacity.

Protein denaturation occurs when baking bread, drying pasta, vegetables, during cooking, etc. As a result, the biological value of these proteins increases, since denatured (partially destroyed) proteins are more easily absorbed during digestion.

Isoelectric point of a protein. Proteins contain various basic and acidic groups that have the ability to ionize. In a strongly acidic medium, the main groups (amino groups, etc.) are actively protonated, and protein molecules acquire a total positive charge, and in a strongly alkaline medium, carboxyl groups easily dissociate, and protein molecules acquire a total negative charge.

The sources of a positive charge in proteins are the side radicals of lysine, arginine and histidine residues, and the a-amino group of the N-terminal amino acid residue. The sources of the negative charge are the side radicals of aspartic and glutamic acid residues, and the α-carboxyl group of the C-terminal amino acid residue.

At a certain pH value of the medium, the equality of positive and negative charges on the surface of the protein molecule is observed, i.e., its total electric charge turns out to be zero. This pH value of the solution, at which the protein molecule is electrically neutral, is called the isoelectric point of the protein (pi).

Isoelectric points are characteristic constants of proteins. They are determined by their amino acid composition and structure: the number and arrangement of acidic and basic amino acid residues in polypeptide chains. The isoelectric points of proteins, in which acidic amino acid residues predominate, are located in the pH region.<7, а белков, в которых преобладают остатки основных аминокислот - в области рН>7. The isoelectric points of most proteins are in a slightly acidic environment.

In the isoelectric state, protein solutions have a minimum viscosity. This is due to a change in the shape of the protein molecule. At the isoelectric point, oppositely charged groups are attracted to each other, and the proteins twist into balls. When the pH shifts from the isoelectric point, like-charged groups repel each other, and the protein molecules unfold. In the unfolded state, protein molecules give solutions a higher viscosity than rolled into balls.

At the isoelectric point, proteins have minimal solubility and can easily precipitate.

However, precipitation of proteins at the isoelectric point still does not occur. This is prevented by structured water molecules that retain a significant portion of hydrophobic amino acid radicals on the surface of protein globules.

Proteins can be precipitated using organic solvents (alcohol, acetone), which disrupt the system of hydrophobic contacts in protein molecules, as well as high salt concentrations (salting out), which reduce the hydration of protein globules. In the latter case, part of the water goes to dissolve the salt and ceases to participate in the dissolution of the protein. Such a solution, due to a lack of solvent, becomes supersaturated, which entails the precipitation of part of it in the precipitate. Protein molecules begin to stick together and, forming ever larger particles, gradually precipitate out of solution.

Optical properties of a protein. Solutions of proteins have optical activity, i.e., the ability to rotate the plane of polarization of light. This property of proteins is due to the presence of asymmetric elements in their molecules - asymmetric carbon atoms and a right-handed a-helix.

When a protein is denatured, its optical properties change, which is associated with the destruction of the a-helix. The optical properties of fully denatured proteins depend only on the presence of asymmetric carbon atoms in them.

By the difference in the manifestation of the optical properties of the protein before and after denaturation, one can determine the degree of its spiralization.

Qualitative reactions to proteins. Proteins are characterized by color reactions due to the presence of certain chemical groups in them. These reactions are often used to detect proteins.

When copper sulfate and alkali are added to the protein solution, a lilac color appears, associated with the formation of complexes of copper ions with peptide groups of the protein. Since this reaction gives biuret (H 2 N-CO-NH-CO-NH 2), it is called biuret. It is often used for the quantitative determination of protein, along with the I. Kjeldahl method, since the intensity of the resulting color is proportional to the protein concentration in the solution.

When protein solutions are heated with concentrated nitric acid, a yellow color appears due to the formation of nitro derivatives of aromatic amino acids. This reaction is called xantoprotein(Greek "xanthos" - yellow).

Many protein solutions, when heated, react with a nitrate solution of mercury, which forms raspberry-colored complex compounds with phenols and their derivatives. This is a qualitative Millon test for tyrosine.

As a result of heating most protein solutions with lead acetate in an alkaline medium, a black precipitate of lead sulfide precipitates. This reaction is used to detect sulfur-containing amino acids and is called the Fohl reaction.