Xantoprotein reaction to protein: signs and formula of the equation

To establish the qualitative composition of many food products, a xantoprotein reaction to protein is used. The presence of aromatic amino acids in the compound will give a positive color change to the sample under study.

What is protein

It is also called a protein, which is a building material for a living organism. Proteins support the volume of muscles, restore the traumatized and dead tissue structures of various organs, whether it be hair, skin or ligaments. With their participation, red blood cells are produced, normal functioning of many hormones and cells of the immune system is regulated.

This is a complex molecule, which is a polypeptide with a mass greater than 6 * 10 ³ daltons. The protein structure is formed by amino acid residues in large amounts, connected by a peptide bond.

Structure of proteins

A distinctive feature of these substances in comparison with low-molecular peptides is their developed spatial three-dimensional structure, supported by influences with different degrees of attraction. Proteins have a four-level structure. For each of them, their characteristics are inherent.

The basis of the primary organization of their molecules is an amino acid sequence, the structure of which recognizes the xantoprotein reaction to protein. Such a structure is a periodically repeating peptide bond -HN-CH-CO-, and the selective part is the side chain radicals in the aminocarboxylic acids. They determine the properties of the substance as a whole in the future.

The primary protein structure is considered quite strong, this is due to the presence of strong covalent interactions in peptide bonds. The formation of subsequent levels occurs depending on the signs established at the initial stage.

The formation of the secondary structure is possible due to the twisting of the amino acid sequence into a helix in which hydrogen bonds are established between the turns.

The tertiary level of the organization of the molecule is formed when one part of the helix is superimposed on other fragments with the appearance of all possible bonds between them, with hydrogen, disulphide, covalent or ionic compounds. As a result, associations are obtained in the form of globules.

The spatial arrangement of tertiary structures with the formation of chemical bonds between them leads to the formation of a finite species of the molecule or a quaternary level.

Amino acids

They are responsible for the chemical properties of proteins. There are about 20 main amino acids that make up the polypeptides in different sequences. This also includes rare aminocarboxylic acids in the form of hydroxyproline and hydroxylizine, which are derived from basic peptides.

As a sign of a xantoprotein protein recognition reaction, the presence of individual amino acids gives a change in the color of the reagents, suggesting the presence of specific structures in their composition.

As it turned out, all of them are carboxylic acids, in which there was a substitution of the hydrogen atom for the amino group.

An example of the structure of a molecule is the structural formula of glycine (HNH-HCH-COOH) as the simplest amino acid.

In this case, one of the CH ₂ -carbon hydrogens can be replaced by a longer radical including the benzene ring, amino, sulfonic, carboxy groups.

What does the xantoprotein reaction mean?

To carry out qualitative analysis of proteins, various methods are used. They include reactions:

• Biuretovuyu with the appearance of violet staining;
• Ninhydrino to form a blue-violet solution;
• Formaldehyde with the establishment of red coloration;
• Fole with precipitation of gray-black color.

In carrying out each method, the presence of proteins and the presence of a certain functional group in their molecule are demonstrated.

There is a xantoprotein reaction to the protein. It is also called Mulder's breakdown. It refers to color reactions to proteins in which aromatic and heterocyclic amino acids are present.

A feature of such a sample is the process of nitration with an acid of a nitrogen cyclic amino acid residue, in particular, the addition of a nitro group to a benzene ring.

The result of this process is the formation of a nitro compound that precipitates. This is the main sign of the xantoprotein reaction.

What are the amino acids

Not all aminocarboxylic acids can be detected by such a sample. The main feature of the xantoprotein protein recognition reaction is the presence of a benzene ring or heterocycle in the amino acid molecule.

Of the protein amino carboxylic acids, two aromatic acids are distinguished, in which there is a phenyl group (in phenylalanine) and a hydroxyphenyl radical (in tyrosine).

Using a xantoprotein reaction, a heterocyclic amino acid tryptophan is determined having an aromatic indole nucleus. The presence of the above compounds in the protein gives a characteristic change in the color of the test medium.

What reagents are used

To carry out the xantoprotein reaction, it will be necessary to prepare a 1% solution of egg or vegetable protein.

Usually a chicken egg is used, which is broken to further separate the protein from the yolk. To obtain a solution, 1% protein is diluted in a tenfold amount of purified water. After dissolving the protein, the resulting liquid must be filtered through several layers of gauze. This solution should be stored in a cool place.

It is possible to carry out a reaction with vegetable protein. To prepare the solution, wheat flour is used in an amount of 0.04 kg. Add 0.16 l of purified water. The ingredients are mixed in a flask, which is placed for 24 hours in a cold place with a temperature of about + 1 ° C. After a day, the solution is shaken, after which it is filtered first with cotton wool and then with a paper fold filter. The resulting liquid is kept in a cool place. In such a solution, there is mainly an albumin fraction.

To carry out the xantoprotein reaction, concentrated nitric acid is used as the main reagent. Additional reagents are a solution of 10% sodium hydroxide or ammonia, a gelatin solution and an unconcentrated phenol.

Methods of conducting

A 1% solution of egg or flour protein in an amount of 2 ml is added to a clean test tube. About 9 drops of concentrated nitric acid are added to it, so that the flocculation stops. The resulting mixture is heated, resulting in a yellow precipitate and gradually disappears, and its color turns into a solution.

When the liquid cools down, about 9 drops of sodium hydroxide is added to the tube by stenochka, which is an excess for the process. The reaction of the medium becomes alkaline. The contents in the test tube become orange in color.

Features

Since xantoprotein is called a qualitative reaction to proteins under the action of nitric acid, the sample is taken under the enclosed hood. Observe all safety precautions when handling concentrated caustic substances.

During the heating process, the contents can be ejected from the tube, which should be taken into account when attaching it to the holder and selecting the slope.

Recruit concentrated nitric acid and caustic sodium only with a glass pipette and a rubber pear, it is forbidden to pick up your mouth.

Comparative reaction with phenol

For clarity of the process and confirmation of the presence of a phenyl group, a similar test with hydroxybenzene is carried out.

In a test tube, add 2 ml of phenol diluted, then gradually, by stenochke, add 2 ml of acid nitric concentrated. The solution is subjected to heating, whereby it becomes yellow. This reaction is qualitative for the presence of a benzene ring.

The process of nitration of hydroxybenzene with nitric acid is accompanied by the formation of a mixture of paranitrophenol and orthotriphenol in a percentage ratio of 15 to 35.

Comparative test with gelatin

To prove that the xantoprotein reaction to a protein reveals amino acids only with an aromatic structure, proteins that do not have a phenolic group are used.

A 1% gelatin solution in a quantity of 2 ml is added to a clean test tube. Approximately 9 drops of concentrated nitric acid are added to it. The resulting mixture was heated. The solution is not colored yellow, which proves the absence of amino acids with an aromatic structure. Sometimes there is a slight yellowing of the medium, due to the presence of protein impurities.

Chemical equations

The xantoprotein reaction to proteins takes place in two stages. The formula of the first stage describes the process of nitration of an amino acid molecule with a concentrated nitrogenous acid.

An example is the addition of a nitro group to the tyrosine with the formation of nitrotyrosine and dinitrotyrosine. In the first case, one NO ₂ -radical is attached to the benzene ring, and in the second compound already two hydrogen atoms are replaced by NO ₂ . The chemical formula of the xantoprotein reaction is represented by the interaction of tyrosine with nitric acid with the formation of a nitrotyrosine molecule.

The nitration process is accompanied by a transition of colorless coloration to a yellow tone. When this reaction is carried out with proteins containing amino acid residues of tryptophan or phenylalanine, the color of the solution also changes.

At the second stage, the nitration products of the tyrosine molecule, in particular nitrotrozine, are reacted with ammonium or sodium hydroxide. The result is a sodium or ammonium salt, in which the color is yellow-orange. Such a reaction is connected with the possibility of a molecule of nitrothyrosine to pass into a quinoid form. Later it forms a salt of nitronic acid, which has a quinone system of double conjugated bonds.

Thus, the xantoprotein reaction to proteins ends. The equation of the second stage is presented above.

results

In the process of analyzing the liquids contained in three test tubes, the dilution phenol serves as the reference solution. Substances with a benzene ring give a qualitative reaction with a nitric acid. As a result, the color of the solution changes.

As is known, gelatin includes collagen in a hydrolysed form. This protein does not contain aminocarboxylic acids of aromatic structure. When interacting with an acid, the color of the medium does not change.

In the third test tube, a positive xantoprotein reaction to proteins is observed. The conclusion can be made the following: all proteins with an aromatic structure, be it a phenyl group or an indole ring, give a color change of the solution. This is due to the formation of nitro compounds with a yellow color.

Conducting a color reaction proves the presence of a diverse chemical structure in amino acids and proteins. An example with gelatin shows that it contains aminocarboxylic acids that do not have a phenyl group or a cyclic structure.

Using the xantoprotein reaction, it is possible to explain the yellowing of the skin when strong nitric acid is applied to it. The same color will be acquired by milk foam when carrying out similar analysis with it.

In medical laboratory practice, this color sample is not used to detect protein in the urine. This is due to the yellow coloration of the urine itself.

The xantoprotein reaction has become increasingly used to quantitatively count amino acids, such as tryptophan and tyrosine, in various proteins.