1) Biuret reaction(for all proteins)
Protein + CuSO 4 + NaOH bright purple color
СuSO 4 + 2NaOH Cu(OH) 2 + Na 2 SO 4
blue sediment
C = O: Cu: O = C C = O: N
N H OH N: O = C
soluble complex
bright purple
2) Xanthoprotein reaction(for proteins containing AA with an aromatic radical)
protein + HNO 3 (k) yellow precipitate
| || -- H 2 O | ||
N CH C─ + HONO 2 N CH C─
|
yellow
If you add a concentrated ammonia solution, an orange color appears because the electron density shifts in nitrobenzene.
3) Cysteine reaction- reaction to an AK residue containing S
Protein + NaOH + Pb(CH 3 COO) 2 PbS + protein
Black
| Pb + PbS
BIOCATALYSIS
One of important features chemical reactions occurring in living organisms is their catalytic nature. Living cell can be thought of as a miniature catalytic reactor. The difference between a cell and a chemist’s flask is that if in a flask all reactions proceed independently (the fundamental principle of independence of reactions is implemented), then in a cell everything happens interconnected.
This does not happen because they are violated physical laws or does the cell obey other laws - no, only laws operate in living matter. It’s just that in the process of evolution, nature created an effective apparatus for regulating all cellular reactions, which allows the entire cell to control the ratio of products in such a way that all reactions function optimally.
So everything is bio chemical reactions- these are reactions catalytic.
Biological catalysts are called enzymes or enzymes.
In principle, the same chemical reactions take place in the cell as in a chemical laboratory, but strict restrictions are imposed on the conditions for the reactions in the cell, namely T = 37 ◦ C and P = 1 atm.
Therefore, often processes that occur in one stage in the laboratory are carried out in several stages in living cells.
The essence of catalytic reactions, despite their diversity, boils down to the fact that the starting materials form with the catalyst intermediate connection, which relatively quickly turns into reaction products, regenerating the catalyst.
Sometimes intermediates can be isolated in pure form, but usually they consist of unstable molecules that can only be detected using very sensitive spectral instruments.
The process involving a catalyst is cyclic or circular.
A measure of enzyme activity - speed(number of moles of substrate undergoing a change in 1 minute per 1 mole of enzyme)
The number of revolutions can reach 10 8.
Quite often, the cycles of several catalysts are combined together, forming a circular process.
Substances S1 and S2 are converted into products P1 and P2. During this transformation, first S1 reacts with a third substance X and catalyst E1, forming intermediate product M1, which in turn is converted by catalyst E2 into intermediate product M2, etc.
The accelerating effect of a catalyst is associated with a decrease in activation energy (this is the additional energy that must be imparted to one mole of a substance in order for the particles of the substance to become reactive and able to overcome the energy barrier of the reaction).
The main properties of enzymes include:
Efficiency, which lies in the degree of acceleration (acceleration by 100 million times).
Increased substrate specificity. Enzymes distinguish the substrate through biological recognition (complementarity).
Increased specificity of the catalyzed reaction. Most enzymes speed up one type of reaction.
Increased specificity for optical isomers (can recognize left-handed and right-handed isomers).
The reason for all the unique properties of enzymes is their spatial structure. Typically these are globular proteins, much larger than the substrate in size. This circumstance leads to the fact that in the process of evolution an active center was formed on the surface of the enzyme, which is complementary to the substrate. This is a lock and key.
Conditionally active centers are divided into: binding and catalytic.
The binding center binds the substrate and optimally orients it in relation to the catalyzed group, while all active groups are concentrated in the catalytic center.
If hydrolysis (of proteins, lipids) is necessary to carry out a reaction, then the catalyzed center is formed by side radicals of AA residues.
In this case, the enzyme consists only of polypeptide chains. However, in addition to hydrolytic reactions, others also occur: redox reactions, transfer reactions of any groups.
In these cases, the enzymes contain a non-protein part. This part is coenzyme(r-factor, prosthetic group). The protein part provides the binding effect, and the coenzyme provides the catalytic effect. Protein part - apoenzyme.
Apoenzyme + coenzyme ↔holoenzyme
Denaturation of proteins.
Demonstration of experiments from the presentation “Squirrels”:
Coagulation of proteins when heated Precipitation of proteins by salts of heavy metals
Protein precipitation with alcohol
Teacher. Proteins are characterized by reactions that result in the formation of a precipitate. But in some cases, the resulting precipitate dissolves with excess water, and in others, irreversible protein coagulation occurs, i.e. denaturation. Renaturation- This is the reverse process of denaturation.
What can denaturation lead to?
Impaired antigenic sensitivity of the protein;
Blocking a number of immunological reactions;
Metabolic disorders;
Inflammation of the mucous membrane of a number of digestive organs (gastritis, colitis);
Stone formation (stones have a protein base).
Conclusion: Denaturation of proteins– a complex process in which, under the influence of external factors: temperature, the action of chemical reagents, mechanical stress and a number of others, a change occurs in the secondary, tertiary and quaternary structures of the protein macromolecule. The primary structure, and therefore chemical composition the protein does not change. During denaturation they change physical properties protein, solubility decreases, biological activity is lost, the shape of the protein macromolecule changes, and aggregation occurs.
Protein hydrolysis (from the chapter “ 8. Chemical properties proteins").
Teacher. Protein hydrolysis- This is, first of all, the destruction of one of the most important levels of organization of the protein molecule. Protein hydrolysis- destruction of the primary protein structure under the action of acids, alkalis or enzymes, leading to the formation of α-amino acids from which it was composed.
Color reactions to proteins (biuret)
Biuret reaction
Teacher. Biuret reaction– reaction to peptide bonds.
Protein + Cu(OH) 2 → violet color of the solution
In addition to the biuret reaction, there are a number of color reactions that make it possible to prove the presence of individual fragments of a protein molecule, for example xanthoprotein.
Demonstration of experience from the presentation “Squirrels”:
Xanthoprotein reaction
Teacher. Xanthoprotein reaction- reaction to aromatic cycles.
Protein + HNO 3 (k) → white precipitate → yellow color → orange color
Proteins burn to produce nitrogen, carbon dioxide and water, as well as some other substances. Combustion is accompanied by the characteristic smell of burnt feathers.
Proteins undergo decay (under the influence of putrefactive bacteria), which produces methane (CH 4), hydrogen sulfide (H 2 S), ammonia (NH 3), water and other low-molecular products.
CONCLUSION:
PROTEINS– biopolymers of irregular structure, the monomers of which are 20 amino acids of different types. The chemical composition of amino acids includes: C, O, H, N, S. Protein molecules can form four spatial structures and perform a number of functions in the cell and body: construction, catalytic, regulatory, motor, transport, etc.
Squirrels- the basis of life on Earth, are part of the skin, muscle and nerve tissue, hair, tendons, walls of blood vessels of animals and humans; it is the building material of the cell. The role of proteins can hardly be overestimated, i.e. life on our planet can really be considered as a way of existence of protein bodies that exchange substances and energy with the external environment.
Since protein contains a variety of functional groups, it cannot be classified into any of the previously studied classes of compounds. It combines, like a focal point, the characteristics of compounds belonging to different classes. This, combined with the features of its structure, characterizes the protein as higher form development of matter.
You can quote the words of L. Pauling: “With good reason we can say that proteins are the most important of all substances that make up the organisms of animals and plants.”
Presentation demonstration "Squirrels"-CONCLUSIONS Statements about the life and proteins of famous people
people
“Wherever we find life, we find it associated with some protein body.”
proteins from which ... are built.
In the structure of a protein, there are... structures.
Functions of proteins in the body...
Proteins; α-amino acid residues.
S, N, O, N, S.
Ten thousand, millions.
Water, solutions of salts, acids; alkalis.
Tissues of living organisms: skin, tendons, muscles, nails, hair.
Primary, secondary, tertiary, quaternary.
Construction, catalytic, propulsion, transport, protective, energy.
Evaluation criteria:
"5" - all answers are correct; "3" - 3 incorrect answers;
"4" - 1-2 incorrect answers; "2" - 4 or more incorrect answers.
Basic summary:
Proteins are complex high-molecular natural compounds built from α - amino acid residues connected by peptide (amide) bonds - CO - NH -.
The number of amino acid residues included in protein molecules is different: insulin - 51, myoglobin - 140. Mr (protein) = from 10,000 to several million.
Mr (egg white) = 36,000; Mr (muscle protein) = 1,500,000.
Hemoglobin (C738H1166O208N203S2Fe) 4.
Protein structures.
Primary- sequence of alternation of amino acid residues (all bonds are covalent, strong).
Secondary- form polypeptide chain in space (most often a spiral). The protein chain is twisted into a spiral (due to many hydrogen bonds). Tertiary- the real three-dimensional configuration that a twisted helix takes in space (due to hydrophobic bonds), some have S - S - bonds (bisulfide bonds).
Quaternary- protein macromolecules connected to each other.
Chemical properties
1) hydrolysis(when heated with solutions of acids, alkalis, under the action of enzymes)
H2N ― CH2 ― C ―: N ― CH ― C ―: N ― CH ― C = O → H2N ― CH2 ― C = O +
H2O CH2 H2O CH2 OH OH
| | glycine
tripeptide
H2N – CH – C = O + H2N – CH – C = O
serine cysteine
Protein hydrolysis is reduced to the hydrolysis of polypeptide bonds. The digestion of proteins also comes down to this:
protein ↔ amino acids → blood into all cells and tissues of the body.
2) denaturation - disruption of the natural structure of the protein (under the influence of heat and chemical reagents)
3) amphotericity:
Properties of acids
|__________ properties of bases
4) protein color reactions - qualitative reactions
a) xanthoprotein reaction.
Protein + HNO3 conc. → yellow color
b) biuret reaction.
Protein + Cu (OH) 2 ↓ → purple solution.
c) combustion- the smell of burnt feathers.
Conclusion: high-quality reactions for proteins are those with concentrated nitric acid(yellow color), with freshly precipitated copper (II) hydroxide (purple solution) and burning of proteins (smell of burnt feathers).
The role of proteins in the cell.
1. Construction material- formation of the cell membrane, organelles and membranes. Blood vessels, tendons, and hair are built.
2. Catalytic role - all cellular catalysts are proteins.
3. Motor function - contractile proteins cause any movement.
4. Transport function - the blood protein hemoglobin attaches oxygen and distributes it to all tissues.
5. Protective role - the production of protein bodies of antibodies to neutralize foreign substances.
6. Energy role: 1 g of protein → 17.6 kJ.
Guidelines for teachers
2. Questions on chemistry to prepare for the seminar must be given to students no later than two weeks before the lesson.
4. The chemistry teacher provides motivation for the lesson, considers the composition and properties of proteins. A biology teacher generalizes and updates knowledge about the structure of protein molecules, their functions and applications.
5. At the end of the lesson, teachers evaluate the students’ work in this lesson. Equipment: code films, overhead projector, screen, overhead projector, slides, chemicals, demonstration table, tables.
Lesson plan (written on the board)
1. Composition and structure of protein.
2. Protein properties (denaturation, renaturation, hydrolysis, color reactions).
3. Functions of protein and its synthesis in the cell.
4. Application of protein, artificial synthesis of peptides.
Chemistry teacher. Today we are conducting an unusual lesson - it covers the problems of chemistry and biology at the same time. The purpose of our lesson is to systematize and deepen knowledge on the topic “Protein”. We pay special attention to the study of proteins, because proteins are the main component of all life on Earth. Remember F. Engels’ statement about what life is: “Wherever we meet life, we find that it is associated with some kind of protein body, and wherever we find any protein body that is not in the process of decomposition , we, without exception, encounter the phenomena of life. Life is a way of existence of protein bodies.” No substance performs such specific and diverse functions in the body as protein.
Let's remember what compounds are called proteins. ( Natural polymers whose monomers are amino acids.)
The study of which process helped to establish the structure of proteins? ( Study of protein hydrolysis.)
What process is called hydrolysis?
What compounds are formed during the hydrolysis of proteins?
What compounds are called amino acids?
How many amino acids are known in nature?
How many amino acids are found in proteins?
A chemistry teacher demonstrates a code film.
Chemistry teacher. Pay attention to the position of the amino group in amino acids. In accordance with the position of the amino group, the amino acids that make up proteins are called a-amino acids. The general formula of any of these amino acids can be written as follows:
On the code film you see two amino acids, one of which contains two carboxyl groups – COOH, the other – two amino groups – NH2. Such acids are called aminodicarboxylic or diaminocarboxylic acids, respectively.
From your chemistry course you know about optical isomers of natural compounds. Almost all proteins contain only L-amino acids.
Amino acids are monomers of proteins. They can connect to each other through an amide (peptide) bond, which is formed with the release of water - this is a condensation reaction.
Let's create an equation for the reaction between the amino acids glycine and alanine.
(Students work independently and then compare their results with the writing on the board or tape.)
The resulting structure is called a dipeptide. A polymer of many amino acids is called a polypeptide.
Biology teacher. Let's continue studying the properties of proteins, but first we'll answer the following questions.
1. How can we explain the diversity of proteins that exists in nature? ( Differences in the composition of amino acids and their different sequence in the polypeptide chain.)
2. What are the levels of organization of a protein molecule? ( Primary – amino acid sequence; secondary – a -spiral or b - folded structure of chain sections; tertiary - the spatial structure of the protein, formed due to the interaction of amino acid residues of remote sections of the chain: a globule for globular proteins, a filamentous structure for fibrillar proteins; quaternary - the union of two or more separate protein molecules.)
3. What type of bond occurs between amino acids in the primary structure? What is another name for this connection? ( Covalent bond. Amide or peptide bond.)
4. What bonds mainly provide the secondary structure of a protein molecule? ( Hydrogen bonds, disulfhydryl bridges.)
5. What connections provide tertiary structure? ( Hydrogen bonds, hydrophobic and ionic interactions.)
6. What bonds provide the quaternary structure of a protein molecule? ( Electrostatic, hydrophobic and ionic interactions.)
7. Give an example of a protein known to you that has a quaternary structure. ( ATPase, hemoglobin.)
Now let's solve the following problem ( the condition of the task is projected through an overhead projector, a slide is shown with blood smears of a healthy person and a patient with sickle cell anemia).
The disease sickle cell anemia is accompanied by the replacement of the amino acid residue glutamic acid in the polypeptide chain of the hemoglobin molecule with a valine residue. Fragment of the chain of normal hemoglobin: – glu–glu–Liz–. Fragment of an abnormal hemoglobin chain: – shaft–glu–Liz–
(glu– glutamic acid; Liz– lysine; shaft– valine). Draw these fragments as chemical formulas.
Solution.
Fragment of a chain of normal hemoglobin:
Fragment of an abnormal hemoglobin chain:
From the above example it follows that primary structure of a protein molecule can determine all its subsequent levels of organization. Changes in the structural organization of a protein can disrupt its functions, which in some cases leads to the development of pathology - disease.
The structure of a protein determines its physicochemical properties, such as solubility.
A chemistry teacher demonstrates a code film.
Classification of proteins according to their solubility
Chemistry teacher. To maintain their functional activity, proteins must have a natural (native) structural organization at all levels.
Disturbances in the primary organization, leading to the rupture of the amide bond with the addition of a water molecule, are called protein hydrolysis. With complete hydrolysis, the protein breaks down into its constituent amino acids.
Violation of the secondary and tertiary structure of the protein, i.e. the loss of its native structure is called protein denaturation.
Protein denaturation is caused by various factors: significant changes in temperature, increasing and decreasing pH of the environment, exposure to heavy metal ions, some chemical compounds, for example, phenols.
A chemistry teacher demonstrates experiments.
Experience 1. Protein + heat -->
Experience 2. Protein + phenol --> denaturation (precipitation).
Experience 3. Protein + Pb or CH 3 COOH --> denaturation (precipitation).
Experience 4. Protein + CuSO4 --> denaturation (precipitation).
Biology teacher. Denaturation occurs as a result of the destruction of hydrogen and disulfide covalent bonds(but not peptide bonds, ionic and hydrophobic interactions), which ensure the formation and maintenance of the secondary and tertiary structures of the protein. In this case, the protein loses its inherent biological properties.
Reactions used to determine the composition of a substance are called qualitative.
What reactions are qualitative to protein?
A chemistry teacher demonstrates the following experiments.
Experience 1. Xanthoprotein reaction (nitration of benzene rings of aromatic amino acids of protein):
protein (cooled) + HNO 3 (conc.) + heat --> yellow color
Experience 2. Biuret reaction (allows you to determine the number of peptide bonds):
protein + CuSO 4
+ NaOH --> violet color (urea gives this reaction);
CuSO 4
+ NaOH --> Cu(OH) 2
+Na 2
SO 4
;
protein + Cu(OH) 2
--> violet coloring.
Is it possible to recognize glycerol, protein, and glucose using one reagent? Can! This reagent is copper hydroxide, it gives different colors to solutions of these substances:
a) glycerol + Cu(OH) 2
--> bright blue solution;
b) glucose + Cu(OH) 2
+ heating --> red precipitate;
c) protein + Cu(OH) 2
--> violet coloring.
Biology teacher. Name the functions of polypeptides that you know. ( Construction Polypeptides are part of the cell walls of fungi and microorganisms and are involved in the construction of membranes. Hair, nails, and claws are made of keratin protein. Collagen protein is the basis of tendons and ligaments. Another important function of protein is enzymatic, catalytic.
Proteins also provide all types of biological mobility. In addition, proteins perform transport, hormonal, or regulatory, receptor, hemostatic, toxigenic, protective and energy functions.)
Define enzymes. ( Enzymes are proteins that have catalytic activity, i.e. accelerating reactions.)
All enzymes are highly specific to their substrate and, as a rule, catalyze only one very specific reaction. Look at the diagrammatic representation of the structure of an enzyme. ( A biology teacher demonstrates a code film with a schematic representation of an enzyme.) Each enzyme has an active site in which the chemical transformation of the reaction substrate occurs. Sometimes there may be several substrate binding sites. The structure of the binding site is complementary to the structure of the substrate, i.e. they fit together “like a key fits a lock.”
The work of enzymes is influenced by numerous factors: pH, temperature, ionic composition of the medium, the presence of small organic molecules, which bind to the enzyme or are part of its structure and are otherwise called cofactors (coenzymes). Some vitamins, such as pyridoxine (B 6
) and cobalamin (B 12
).
A biology teacher introduces students to the practical use of enzymes.
Clinical significance of enzymes
1. Diseases caused by enzyme deficiency are widely known. Examples: indigestibility of milk (no lactase enzyme); hypovitaminosis (vitamin deficiency) – the lack of coenzymes reduces enzyme activity (hypovitaminosis of vitamin B1 leads to beriberi disease); phenylketonuria (caused by a violation of the enzymatic conversion of the amino acid phenylalanine to tyrosine).
2. Determination of enzyme activity in biological fluids has great value for diagnosing diseases. For example, viral hepatitis is determined by the activity of enzymes in the blood plasma.
3. Enzymes are used as reagents in the diagnosis of certain diseases.
4. Enzymes are used to treat certain diseases. Examples of some enzyme-based drugs: pancreatin, festal, lidase.
Use of enzymes in industry
1. In the food industry, enzymes are used in the preparation of soft drinks, cheeses, canned food, sausages, and smoked meats.
2. In animal husbandry, enzymes are used in the preparation of feed.
3. Enzymes are used in the production of photographic materials.
4. Enzymes are used in the processing of flax and hemp.
5. Enzymes are used to soften leather in the leather industry.
6. Enzymes are part of washing powders.
Biology teacher. Let's look at other functions of proteins. Motor functions are carried out by special contractile proteins, which include, for example, actin and myosin, which are part of muscle fibers.
Another important function of proteins is transport. Proteins, for example, carry potassium ions, amino acids, sugars and other compounds across the cell membrane into the cell. Proteins are also interstitial carriers.
By regulating the metabolism within cells and between cells and tissues of the whole body, proteins perform a hormonal, or regulatory function. For example, the hormone insulin is involved in the regulation of both protein and fat metabolism.
On the surface of cell membranes there are protein receptors that selectively bind hormones and mediators, thereby performing a receptor function.
The homeostatic function of proteins is to form a clot when stopping bleeding.
Some proteins and peptides released by organisms, such as pathogens or some poisonous animals, are toxic to other living organisms - this is the toxicogenic function of proteins.
The protective function of proteins is very important. Antibodies are proteins that are produced by the body's immune system when it is invaded by a foreign protein, bacteria, or virus. They identify the “stranger” and participate in his destruction.
Proteins that serve as an energy reserve include, for example, casein, the main protein in milk.
Answer the following questions.
2. What causes the rejection of transplanted organs and tissues in patients? ( Antibodies, performing a protective function, recognize the foreign protein of the transplanted organs and cause reactions of its rejection.)
3. Why do boiled eggs never produce a chicken? ( Egg whites have irreversibly lost their native structure due to heat denaturation.)
4. Why does the weight of meat and fish decrease after cooking? ( During heat treatment, denaturation of meat or fish proteins occurs. Proteins become practically insoluble in water and give up a significant part of the water they contain, while the weight of meat decreases by 20–40%.)
5. What does the formation of “flakes” or cloudiness of the broth indicate when cooking meat? ( If meat is immersed in cold water and heated, soluble proteins from the outer layers of the meat are transferred into the water. During cooking, they denature, resulting in the formation of flakes, foam that floats to the surface of the water, or a fine suspension that makes the solution cloudy.)
All protein molecules have a finite lifespan - they break down over time. Therefore, proteins are constantly renewed in the body. In this regard, let us recall the basics of protein biosynthesis. Answer the following questions.
1. Where does protein synthesis occur in the cell? ( On ribosomes.)
2. In what cellular organelle information about the primary structure of the protein is stored. ( In chromosomes, the information carrier is DNA.)
3. What is meant by the term “gene”? ( Nucleotide sequence encoding the synthesis of one protein.)
4. What are the main stages of protein biosynthesis called? ( Transcription, broadcast.)
5. What does transcription consist of? ( This is reading information from DNA by synthesizing messenger RNA that is complementary to the DNA region being read.)
6. In what part of the cell does transcription take place? ( In the core.)
7. What does the broadcast consist of? ( This is the synthesis of protein from amino acids in the sequence recorded in mRNA; it occurs with the participation of transport tRNAs that deliver the corresponding amino acids to the ribosome.)
8. In what part of the cell does translation take place? ( In the cytosol, on ribosomes, in mitochondria.)
Protein biosynthesis occurs in the body throughout life, most intensively in childhood. The intensity of protein synthesis in some cases can be adjusted. The action of many antibiotics is based on the suppression of protein synthesis, including in bacteria that cause the disease. For example, the antibiotic tetracycline prevents tRNA from binding to ribosomes.
Let's listen short messages about protein preparations used in modern medicine.
Antihistamines
The modern busy rhythm of life is accompanied by an increase in the number of diseases, such as heart attack, hypertension, obesity, and all kinds of allergies. Allergy is the body's excessive sensitivity to specific external irritants. All these diseases are characterized by increased levels of histamine in the blood. Histamines are substances formed by decarboxylation of the amino acid histidine. Antihistamines interfere with this reaction and histamine levels decrease.
Interferon
In the process of evolution, in the fight against viruses, animals have developed a mechanism for the synthesis of the protective protein interferon. The program for the formation of interferon, like any protein, is encoded in DNA in the cell nucleus and is turned on after the cells are infected with a virus. Cooling, nervous shock, and lack of vitamins in food lead to a decrease in the ability to produce interferon. Currently, interferon preparations for medical purposes are made from leukocytes from donor blood or using genetic engineering. Interferon is used for prevention and treatment viral infections– influenza, herpes, as well as malignant neoplasms.
Insulin
Insulin is a protein consisting of 51 amino acids. It is released in response to increased blood glucose levels. Insulin controls carbohydrate metabolism and causes the following effects:
– increasing the rate of conversion of glucose into glycogen;
– acceleration of glucose transfer through cell membranes in muscles and adipose tissue;
– increased protein and lipid synthesis;
– increasing the rate of synthesis of ATP, DNA and RNA.
Insulin is necessary for life, because it is the only hormone that reduces the concentration of glucose in the blood. Insufficient secretion of insulin leads to a metabolic disorder known as diabetes mellitus. Insulin preparations are obtained from the pancreas of cattle or through genetic engineering.
Chemistry teacher. Insulin was the first protein whose primary structure was deciphered. It took almost 10 years to establish the sequence of amino acids in insulin. Currently, the primary structure of a very large number of proteins, including those of a much more complex structure, has been deciphered.
The synthesis of protein substances was first carried out using the example of two pituitary hormones (vasopressin and oxytocin).
Finally, teachers give students grades for their work in chemistry and biology class.
The significance of color reactions is that they make it possible to detect the presence of protein in biological fluids, solutions and establish the amino acid composition of various natural proteins. These reactions are used for both qualitative and quantitative determination of protein and the amino acids it contains. Some reactions are inherent not only to proteins, but also to other substances, for example, phenol, like tyrosine, gives a pink-red color with Millon's reagent, so carrying out one reaction is not enough to determine the presence of a protein.
There are two types of color reactions: 1) universal - biuret (for all proteins) and ninhydrin (for all A-amino acids and proteins); 2) specific - only to certain amino acids both in the protein molecule and in solutions of individual amino acids, for example, the Foll reaction (for amino acids containing weakly bound sulfur), the Millon reaction (for tyrosine), the Sakaguchi reaction (for arginine), etc.
When carrying out color reactions for proteins and amino acids, you must first compile the following table:
Color reactions to proteins (qualitative reactions)
Color reactions to proteins Experiment 1. Biuret reaction.
Biuret reaction– quality for everything without exception squirrels, as well as products of their incomplete hydrolysis, which contain at least two peptide bonds.
Principle of the method. The biuret reaction is caused by the presence in proteins of peptide bonds (- CO – NH -), which in an alkaline environment form red-violet colored copper salts with copper (II) sulfate complexes. The biuret reaction is also produced by some non-protein substances, for example biuret(NH 2 -CO-NH-CO-NH 2), oxamide(NH 2 CO-CO-NH 2), series amino acids (histidine, serine, threonine, asparagine).
Biuret reaction with glycine
The order of work.
An equal volume of a 10% solution is added to 1 ml of the test 1% protein solution. sodium hydroxide(NaOH) alkali and then 2-3 drops of 1% solution copper sulfate(CuSO 4). diluted, almost colorless solution of copper sulfate.
If the reaction is positive, a purple color with a red or blue tint appears.
Experience 2.Reactionto “weakly bound sulfur”.
Principle of the method. This is a reaction to cysteine and cystine. During alkaline hydrolysis, the “loosely bound sulfur” in cysteine and cystine is quite easily split off, resulting in the formation of hydrogen sulfide, which, reacting with alkali, produces sodium or potassium sulfides. When lead(II) acetate is added, a gray-black precipitate of lead(II) sulfide is formed.
The order of work.
1 ml of undiluted chicken protein is poured into a test tube, 2 ml of 20% sodium hydroxide solution is added. The mixture is carefully boiled (to prevent the mixture from being thrown away).
In this case, ammonia is released, which is detected by the blueness of wet litmus paper brought to the opening of the test tube (do not touch the wall). The slight precipitate that forms dissolves at boiling, and then 0.5 ml of lead(II) acetate solution is added. A gray-black precipitate of lead(II) sulfide is observed:
Chemistry of the reaction:
|
black sediment |
1 ml is poured into a test tube. add 2 ml of undiluted chicken protein. concentrated alkali solution, put several boilers. A solution of sodium plumbite is added to the hot solution - a yellow-brown or black color is formed. (Sodium plumbite is prepared as follows: an alkali solution is added dropwise to 1 ml of lead acetate until the lead hydroxide precipitate that initially forms a precipitate is dissolved).
If a protein molecule contains sulfur-containing amino acids (cystine, cysteine), sulfur is gradually cleaved from these amino acids in the form of an ion in oxidation state – 2, the presence of which is detected by the lead ion, which forms black insoluble lead sulfide with the sulfur ion:
Pb(CH 3 COO) 2 + 2NaOH Pb(OH) 2 + 2 CH 3 COONa,
Pb(OH) 2 + 2NaOH Na 2 PbO 2 + H 2 O,
Na 2 S + Na 2 PbO 2 + 2H 2 O PbS + 4NaOH.
Experiment 3. Xanthoprotein reaction of proteins.
Principle of the method. This reaction is used to detect a-amino acids containing aromatic radicals. Tyrosine, tryptophan, phenylalanine, when interacting with concentrated nitric acid, form nitro derivatives that are yellow in color. In an alkaline environment, the nitro derivatives of these a-amino acids give orange-colored salts. Gelatin, for example, which does not contain aromatic amino acids, does not give a xanthoprotein test.
The order of work.
Add 0.5 ml of concentrated nitric acid to 1 ml of a 10% chicken egg white solution. As a result of protein coagulation, a white precipitate or cloudiness is formed in the contents of the test tube. When heated, the solution and precipitate turn bright yellow. In this case, the precipitate is almost completely dissolved as a result of hydrolysis. After cooling, add 1–2 ml of 20% sodium hydroxide solution (until the solution turns orange).
Let's consider the mechanism of the xanthoprotein reaction at the tyrosine radical:
Reaction chemistry:
Design of the experiment: draw a conclusion and write the reaction equation.
Experiment 4. Adamkiewicz reaction (to the presence of tryptophan in proteins).
Principle of the method. Proteins containing tryptophan give a red-violet color in the presence of glyoxylic and sulfuric acids. The reaction is based on the ability of tryptophan to react in an acidic environment with glyoxylic acid aldehydes (which is an impurity in concentrated acetic acid) to form colored condensation products. The reaction proceeds according to the equation:
Gelatin does not give this reaction, because. it does not contain tryptophan. The color occurs due to the reaction of tryptophan with glyoxylic acid, which is always present in acetic acid as an impurity.
The same reaction to tryptophan can be carried out using formaldehyde instead of acetic acid, a 2.5% solution of concentrated H 2 SO 4. Stir the solution and after 2-3 minutes. add 10 drops of 5% sodium nitrite while shaking. An intense violet color develops, this is the basis principle of the method reactions.
The order of work.
Pour a few drops of undiluted protein into a test tube and add 2 ml. glacial acetic acid and a few drops of glyoxylic acid. The mixture is slightly heated until the precipitate that forms dissolves, cooled and, tilting the test tube strongly, concentrated H 2 SO 4 is carefully poured along the wall so that the two liquids do not mix.
After 5-10 minutes, the formation of a red-violet ring is observed at the interface between the two layers.
Experiment 5. Ninhydrin reaction.
Principle of the method. a-Amino acids react with ninhydrin, forming a blue-violet complex (Ruemann purple), the color intensity of which is proportional to the amount of amino acid. The reaction proceeds according to the following scheme:
Reaction chemistry :
The reaction with ninhydrin is used for visual detection of a-amino acids on chromatograms (on paper, in a thin layer), as well as for colorimetric determination of the concentration of amino acids based on the color intensity of the reaction product.
The product of this reaction contains the radical (R) of the original amino acid, which causes different colors: blue, red, etc. compounds arising from the reaction of amino acids with ninhydrin.
Currently, the ninhydrin reaction is widely used both for the discovery of individual amino acids and for determining their quantity.
The order of work.
1 ml of a 1-10% diluted solution of chicken egg white and 1-2 ml of a 1% solution of ninhydrin in acetone are poured into a test tube. The contents of the test tube are mixed and carefully heated in a water bath for 2-3 minutes until a blue-violet color appears, indicating the presence of protein α -amino acids.
Design of the experiment: draw a conclusion and write the reaction equation.
Experiment 6. Sakaguchi reaction.
Principle of the method. This reaction to the amino acid arginine is based on the interaction of arginine with a-naphthol in the presence of an oxidizing agent. Its mechanism has not yet been fully elucidated. Apparently, the reaction is carried out according to the following equation:
Since derivatives of quinoneimines (in this case, naphthoquinone), in which the hydrogen of the imino group –NH– is replaced by an alkyl or aryl radical, are always colored yellow-red, then, apparently, the orange-red color of the solution during the Sakaguchi reaction is explained by the appearance of naphthoquinoneimine derivative. However, the possibility of the formation of an even more complex compound due to further oxidation of the remaining NH groups of the arginine residue and the benzene ring of a-naphthol cannot be excluded:
The order of work.
To 2 ml. Add 2 ml of a 1% diluted solution of chicken egg white. 10% sodium hydroxide (NaOH) and a few drops of 0.2% alcohol solution α -naphthol. The contents of the test tube are mixed well. Then add 0.5 ml. sodium hypobromite (NaBrO) or sodium hypochlorite (sodium hypochlorite - NaOCl), mix. A red color immediately appears, gradually intensifying.
Immediately add 1 ml of 40% urea solution to stabilize the rapidly developing orange-red color.
This reaction is typical for compounds containing a guanidine residue
NH = C –NH 2 ,
and indicates the presence of the amino acid arginine in the protein molecule:
NH = C –NH – (CH 2) 3 –CH –COOH
Design of the experiment: draw a conclusion and write the reaction equation.
