The Biuret test is an extensively used procedure to detect the presence of proteins in several body samples like urine, etc. It is also used in colourimetric studies and spectrophotometric analyses involving proteins and peptide complexes
The Biuret Test for Protein is preferred because of its simplicity and ease of availability of the reagent.
What are Proteins?
Proteins are complexes made up of several amino acids linked together at specific locations by a special type of linkage called peptide bonds. To understand the principle behind peptide linkage, one has to be familiar with the structure and properties of amino acids. Basically, an amino acid bears neither a positive nor negative charge. This is because the typical amino acid structure includes:
● A carboxyl group
● An amino group
● A variable side chain.
A peptide bond forms a bridge between the α amino group of amino acid and the α carboxyl group of another amino acid. This is done by the process called condensation. The bonding process is repeated over several cycles to give rise to a polypeptide structure, which is the protein.
The Biuret Reagent
To perform the Biuret Test, the first step is to prepare the Biuret reagent. To prepare a Biuret reagent, NaOH is added to copper sulphate to obtain the alkaline reagent. The reagent is now readily available in almost all laboratories.
The components of the Biuret reagent are as follows:
● Sodium hydroxide or Potassium hydroxide
● Hydrated copper (II) sulphate
● Potassium sodium tartrate
The alkaline nature of the solution is maintained by sodium hydroxide or potassium hydroxide, and the potassium sodium tartrate is mainly present to maintain the stability of the reagent because of its efficient chelating effect.
Materials required for Biuret Test
● Test tubes
● Water bath
● Albumin or egg white preparation
● Deionised or distilled water
● Biuret reagent
Principle of The Biuret Reaction
The peptide bond in itself contains unshared electron pairs from its nitrogen components and from oxygen in the water. These free electrons tend to react with the blue coloured copper sulphate ions when put under an alkaline condition. Upon reaction, these ions form a coloured coordination compound, which causes the collusion to change colour from the blue of the copper sulphate to violet.
The basis of this reaction and colour change is as follows:
● When the copper (II) ions react with the nitrogen from the peptide bond structure, the peptide hydrogens are displaced, provided that the medium is alkaline in nature.
● The four freely available nitrogen atoms provide their electron pairs to the cupric ions and form coordinate covalent bonds. This complex is of chelate type.
● The chelate complex absorbs light at the wavelength of 540 nm, thereby imparting a violet colour to the reacting solution.
The violet colour of the solution can be of varying degrees, ranging from mild to intense violet. This degree of pigmentation is a direct indicator of the number of peptide linkages present in the given protein sample or solution, thereby providing an idea about the total amount of protein present in the solution. More on this is discussed further into the topic.
Procedure for Biuret Test
● Get 3 test tubes. Make sure they are cleaned and dried off properly to avoid unnecessary interactions.
● Label the test tubes appropriately to avoid confusion.
● In test tube 1, add 1 to 2 ml of the given test sample. This is considered the actual test.
● In test tube 2, add 1 to 2 ml of egg albumin. This will be the positive control test tube that will help you confirm your findings later.
● In test tube 3, add 1 to 2 ml of deionised water. This test tube will act as a negative control tube.
● Shake well until the contents are mixed thoroughly.
● Allow the tubes to stand undisturbed for 5 minutes.
● After 5 minutes, observe for changes in the colour of solutions and note down the findings.
Test tubes 1 and 2 show significant changes in the colour of the solution from blue to purple. This is indicative of the presence of peptide bonds and proteins in the given sample solutions. Obtaining a deep violet is indicative of the presence of a large number of proteins in the sample. In contrast, a faded violet colour indicates the presence of a smaller amount of proteins. Tube 3 shows no changes as it was the blank solution.
The results from a Biuret test can either be positive or negative.
● Positive Biuret Reaction: A positive Biuret test is characterised by the significant change in the colour of the solution from copper sulphate blue to violet. All protein and peptide complexes are known to give a positive Biuret test. However, this is not the case for amino acids as such. Histidine is the only amino acid to give a positive Biuret reaction with a noticeable colour change.
● Negative Biuret Reaction: A negative test with Biuret reagent does not produce any colour change because of the absence of peptide linkages in the solution and retains the blue colour of the copper sulphate present previously.
The detection of proteins in samples holds an extremely important therapeutic usage. Protein tests for detecting various body fluids, mainly urine tests, prove very significant because many of the commonly occurring disease conditions have underlying proteinuria.
Proteinuria is nothing but the presence of proteins in the passed urine. It is a significant factor seen in conditions like diabetes mellitus, hypertension (increased blood pressure), and several kidney diseases. With recent advances, the exact value of protein present in a sample can be detected by putting the obtained reaction test tube under a colourimeter or by several other methods. The Biuret test is also used in the procedure for spectrophotometric analysis techniques that are performed to determine the total protein content of a sample solution quantitatively.
● Though the components present in the substances utilised in the test procedure or the reagent are harmless, handle all the materials carefully.
● Make sure to wash hands before and after any procedure to prevent any contamination.
● Double-check the quantity of reagent and samples that you add.
● Maintain a neat and clean workspace and declutter your desk.
● Ensure that the test tubes are cleaned properly and are dried completely. The presence of excess water might interfere with the efficiency of the reaction and affect the overall result.
The Biuret test holds extreme scientific importance, and it is required of any science student to be well-versed in the subject. Proteinuria and associated protein disorders only increase with current progress, which only adds to its crucial role in any laboratory.