To establish the qualitative composition of many food products, a xantoprotein reaction for protein is used. The presence of aromatic amino acids in the compound will give a positive color change to the test sample.
What is protein
It is also called a protein, which is a building material for a living organism. Proteins maintain muscle volume, restore injured and dead tissue structures of various organs, whether it be hair, skin or ligaments. With their participation, red blood cells are produced, the 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 6103 d altons. The protein structure is formed by amino acid residues in large quantities, connected by a peptide bond.
Protein structure
A distinctive feature of these substances in comparison with low-molecular peptides is their developed spatial three-dimensional structure, supported by influences from differentdegree of attraction. Proteins have a four-level structure. Each of them has its own characteristics.
The primary organization of their molecules is based on the amino acid sequence, the structure of which is recognized by the xantoprotein reaction to the protein. Such a structure is a periodically repeating peptide bond -HN-CH-CO-, and the side chain radicals in aminocarboxylic acids are the selective part. It is they who determine the properties of the substance as a whole in the future.
The primary protein structure is considered strong enough, 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 a secondary structure is possible due to the twisting of the amino acid sequence into a spiral, in which hydrogen bonds are established between the turns.
The tertiary level of organization of a molecule is formed when one part of the helix is superimposed on other fragments with the emergence of all kinds of bonds between them, with a hydrogen, disulfide, covalent or ionic compound. The result is associations in the form of globules.
The spatial arrangement of tertiary structures with the formation of chemical bonds between them leads to the formation of the final form of the molecule or the quaternary level.
Amino acids
They determine the chemical properties of proteins. There are about 20 main amino acids,included in the composition of the polypeptides in different sequences. This also includes rare aminocarboxylic acids in the form of hydroxyproline and hydroxylysine, which are derivatives of basic peptides.
As a sign of the xantoprotein reaction of protein recognition, the presence of individual amino acids gives a change in the color of the reagents, which suggests the presence of specific structures in their composition.
As it turned out, they are all carboxylic acids, in which the hydrogen atom was replaced by an 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 hydrogens CH2- carbon can be replaced by a longer radical, including a benzene ring, amino-, sulfo-, carboxy groups.
What does the xantoprotein reaction mean
Different methods are used for qualitative protein analysis. These include reactions:
- biuret with purple coloration;
- ninhydrin to form a blue-violet solution;
- formaldehyde with red staining;
- Foil with gray-black sedimentation.
When carrying out each method, the presence of proteins and the presence of a certain functional group in their molecule are proved.
There is a xantoprotein reaction to protein. It is also called the Mulder test. It refers to color reactions on proteins, inwhich are aromatic and heterocyclic amino acids.
A feature of such a test is the process of nitration of cyclic amino acid residues with nitric acid, in particular, the addition of a nitro group to the benzene ring.
The result of this process is the formation of a nitro compound, which precipitates. This is the main sign of a xantoprotein reaction.
What amino acids are determined
Not all aminocarboxylic acids can be detected using this test. The main feature of the xantoprotein reaction of protein recognition is the presence of a benzene ring or a heterocycle in the amino acid molecule.
From protein aminocarboxylic acids, two aromatic acids are isolated, in which there is a phenyl group (in phenylalanine) and a hydroxyphenyl radical (in tyrosine).
The xantoprotein reaction is used to determine the heterocyclic amino acid tryptophan, which has an aromatic indole nucleus. The presence of the above compounds in the protein gives a characteristic color change of the test medium.
What reagents are used
To carry out the xantoprotein reaction, you will need to prepare a 1% solution of egg or vegetable protein.
Usually use a chicken egg, which is broken to further separate the protein from the yolk. To obtain a solution, 1% protein is diluted in ten times the 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.
You can carry out the reaction with vegetable protein. To prepare the solution, wheat flour is used in the 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 pleated filter. The resulting liquid is kept in a cold 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 non-concentrated phenol.
Methodology
Into a clean test tube add 1% solution of egg protein or flour in the amount of 2 ml. About 9 drops of concentrated nitric acid are added to it to stop the flakes from falling out. The resulting mixture is heated, as a result, the precipitate turns yellow and gradually disappears, and its color goes into solution.
When the liquid cools down, about 9 drops of concentrated sodium hydroxide are added to the test tube along the wall, which is an excess for the process. The reaction of the medium becomes alkaline. The contents in the tube turn orange.
Features
Since xantoprotein is called a qualitative reaction to proteins underby the action of nitric acid, then the test is carried out under the included fume hood. Comply with all safety measures when working with concentrated caustic substances.
During the heating process, the contents of the tube may be ejected, which should be taken into account when fixing it in the holder and choosing an inclination.
Taking concentrated nitric acid and sodium hydroxide should only be done with a glass pipette and a rubber bulb, it is forbidden to type by mouth.
Comparative reaction with phenol
To illustrate the process and confirm the presence of the phenyl group, a similar test is carried out with hydroxybenzene.
Introduce 2 ml of diluted phenol into a test tube, then gradually, along the wall, add 2 ml of concentrated nitric acid. The solution is subjected to heating, as a result of which it turns 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 orthonitrophenol in a percentage ratio of 15 to 35.
Gelatin comparison
To prove that the xantoprotein reaction to a protein only detects amino acids with an aromatic structure, proteins that do not have a phenolic group are used.
Introduce 1% gelatin solution in an amount of 2 ml into a clean test tube. About 9 drops of concentrated nitric acid are added to it. The resulting mixture is heated. The solution does not turn yellow, which proves the absenceamino acids with an aromatic structure. Slight yellowing of the medium is sometimes observed 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 using concentrated nitric acid.
An example is the addition of a nitro group to tyrosine to form nitrotyrosine and dinitrotyrosine. In the first case, one NO2-radical is attached to the benzene ring, and in the second case two hydrogen atoms are replaced by NO2. The chemical formula of the xantoprotein reaction is represented by the interaction of tyrosine with nitric acid to form a nitrotyrosine molecule.
The process of nitration is accompanied by the transition of a colorless color to a yellow tone. When carrying out a similar reaction with proteins containing amino acid residues of tryptophan or phenylalanine, the color of the solution also changes.
At the second stage, the products of nitration of the tyrosine molecule, in particular nitrotyrosine, interact with ammonium or sodium hydroxide. The result is sodium or ammonium s alt, which is yellow-orange in color. This reaction is associated with the ability of the nitrotyrosine molecule to pass into the quinoid form. Later, a s alt of nitronic acid is formed from it, which has a quinone system of double conjugated bonds.
This is how the xantoprotein reaction to proteins ends. Equation Twostages are presented above.
Results
During the analysis of liquids contained in three test tubes, dilute phenol serves as a reference solution. Substances with a benzene ring give a qualitative reaction with nitric acid. As a result, the color of the solution changes.
As you know, gelatin includes collagen in a hydrolyzed form. This protein does not contain aromatic aminocarboxylic acids. When interacting with acid, there is no change in the color of the medium.
In the third test tube, a positive xantoprotein reaction to proteins is observed. The conclusion can be drawn as follows: all proteins with an aromatic structure, be it a phenyl group or an indole ring, give a color change to the solution. This is due to the formation of yellow nitro compounds.
Carrying out a color reaction proves the presence of a variety of chemical structures in amino acids and proteins. The gelatin example shows that it contains aminocarboxylic acids that do not have a phenyl group or a cyclic structure.
The xantoprotein reaction can explain the yellowing of the skin when strong nitric acid is applied to it. Milk froth will acquire the same color when such an analysis is carried out with it.
In medical laboratory practice, this color sample is not used to detect protein in the urine. This is due to the yellow color of the urine itself.
The xantoprotein reaction has become increasingly used to quantify amino acids such as tryptophan and tyrosine in various proteins.
