To establish the qualitative composition of many food products, a xanthoprotein reaction to 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 protein, which is the building material for a living organism. Proteins maintain muscle volume, restore injured and dead tissue structures of various organs, be it 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 of more than 6 * 10 3 daltons. 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 weight peptides is their developed spatial three-dimensional structure, supported by influences with varying degrees 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. They determine in the future the properties of the substance as a whole.
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 depends 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 the molecule is formed when one part of the helix is superimposed on other fragments with the appearance of all kinds of bonds between them, with a hydrogen, disulfide, covalent or ionic compound. The result is an association 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. About 20 major amino acids that make up the polypeptides in a different sequence are counted. Rare aminocarboxylic acids in the form of hydroxyproline and hydroxylysine, which are derivatives of the main peptides, are also included here.
As a sign of a xantoprotein protein recognition reaction, 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, all of them are carboxylic acids, in which the hydrogen atom has been 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 CH 2 - carbon hydrogens can be replaced by a longer radical, including a benzene ring, amino, sulfo, carboxy groups.
What does xantoprotein reaction mean?
To conduct a qualitative analysis of proteins, various methods are used. These include reactions:
- biuret with the appearance of violet staining;
- ninhydrin with the formation of a blue-violet solution;
- formaldehyde with the establishment of red staining;
- Fole with a precipitation of gray-black color.
During each method, the presence of proteins and the presence of a specific 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 to proteins in which aromatic and heterocyclic amino acids are present.
A feature of such a sample is the process of nitration with nitric acid of cyclic amino acid residues, in particular, the attachment of a nitro group to a benzene ring.
The result of this process is the formation of a nitro compound, which precipitates. This is the main sign of xanthoprotein reaction.
What amino acids are determined
Not all aminocarboxylic acids can be detected using such a test. The main sign of a xantoprotein protein recognition reaction is the presence of a benzene ring or heterocycle in the amino acid molecule.
Two aromatic acids are distinguished from protein aminocarboxylic acids in which there is a phenyl group (for phenylalanine) and a hydroxyphenyl radical (for tyrosine).
Using a xantoprotein reaction, a heterocyclic amino acid tryptophan having an aromatic indole core is determined. 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, it will be necessary to prepare a 1% solution of protein of egg or vegetable origin.
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. Such a solution should be stored in a cold place.
You can react 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 folded filter. The resulting liquid is kept in a cold place. In such a solution, mainly the albumin fraction is present.
For carrying out the xantoprotein reaction, concentrated nitric acid is used as the main reagent. Additional reagents are a solution of 10% sodium or ammonia hydroxide, a gelatin solution and non-concentrated phenol.
Methodology
A 1% solution of egg or flour protein in an amount of 2 ml is added to a clean tube. About 9 drops of concentrated nitric acid are added to it to stop the loss of flakes. The resulting mixture is heated, as a result, the precipitate turns yellow and gradually disappears, and its color goes into solution.
When the liquid has cooled, 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 test tube turn orange.
Features
Since xantoprotein is called a qualitative reaction to proteins under the action of nitric acid, the test is carried out under a fume hood. Observe all safety measures when working with concentrated caustic substances.
During heating, the contents may be ejected from the tube, which should be taken into account when attaching it to the holder and choosing a tilt.
Concentrated nitric acid and sodium hydroxide should be collected only with the help of a glass pipette and a rubber pear;
Comparative reaction with phenol
To illustrate the process and confirm the presence of a phenyl group, a similar test with hydroxybenzene is carried out.
2 ml of diluted phenol is added to the test tube, then gradually 2 ml of concentrated nitric acid are added along the wall. The solution is heated, causing it to turn yellow. This reaction is qualitative for the presence of a benzene ring.
The nitration process 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.
Comparison test with gelatin
To prove that the xantoprotein reaction to a protein reveals aromatic amino acids only, proteins that do not have a phenolic group are used.
A 1% gelatin solution in an amount of 2 ml was added to a clean 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 absence of aromatic amino acids. Sometimes there is a slight yellowing of the medium associated with the presence of protein impurities.
Chemical equations
In two stages, a xanthoprotein reaction to proteins takes place. The formula of the first stage describes the process of nitration of an amino acid molecule with concentrated nitric acid.
An example is the attachment of a nitro group to 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 xanthoprotein reaction is represented by the interaction of tyrosine with nitric acid to form a nitrotyrosine molecule.
The nitration process is accompanied by a 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 nitration products of the tyrosine molecule, in particular nitrotyrosine, react with ammonium or sodium hydroxide. The result is a sodium or ammonium salt in which the color is yellow-orange. This reaction is associated with the ability of the nitrotyrosine molecule to go into the quinoid form. Subsequently, a nitronic acid salt is formed from it, which has a quinone system of double conjugated bonds.
So the xantoprotein reaction to proteins ends. The second stage equation is presented above.
results
In the analysis of liquids contained in three test tubes, diluted phenol is used 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 hydrolyzed form. This protein does not contain aromatic aminocarboxylic acids. When interacting with acid, the color of the medium does not change.
In the third tube, a positive xantoprotein reaction to proteins is observed. The conclusion can be drawn as follows: all proteins with an aromatic structure, whether it is a phenyl group or an indole ring, give a color change to the solution. This is due to the formation of nitro compounds with a yellow color.
The color reaction proves the presence of a diverse chemical structure in amino acids and proteins. An example with gelatin shows that aminocarboxylic acids that do not have a phenyl group or a cyclic structure are present in its composition.
With the help of a xanthoprotein reaction, yellowing of the skin can be explained when strong nitric acid is applied to it. Milk foam will get the same color when conducting a 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 color of the urine itself.
The xanthoprotein reaction has become increasingly used for the quantitative calculation of amino acids such as tryptophan and tyrosine in various proteins.