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Biuret Test and its Principle, Preparation and Procedure

Testing the presence of protein can be crucial for multiple food and non-edible components. And one of the tests most suitable to do so is the Biuret test.

Learn the highly effective method of conducting the Biuret method for determination of protein.

Objective

  1. To determine the presence of peptide bonds.
  2. To detect the protein in the given solution.

Principle

  1. The urea is heated to 180° to form a biuret.
  2. The biuret reagent reacts with peptide bonds in protein in an alkaline environment to form violet-coloured biuret complexes. A coloured coordination complex is formed between the peptide bond of carbonyl oxygen (>C=O) and amide nitrogen (=NH) of the amino acids and Cu2  ion.
  3. The intensity of colour depends on the number of peptide bonds in the solution. The deeper the violet colour, the higher is the number of peptide-copper complexes.
  4. An alkaline environment is important to perform this experiment as it helps break the proteins, making it easier for Cu2  ions from Copper sulfate to react with peptides.
  5. The Copper(II) ions react with the valence electrons of 4 nitrogen atoms of the 4 different peptides for a coloured coordinate complex.
  6. Four nitrogen atoms donate lone pairs of electrons to form coordinate covalent bonds with the cupric ions, resulting in a chelate complex. The so-formed chelate complex can absorb light of a wavelength of 540 nm, giving purple colour. Therefore, the formation of a purple-coloured complex indicates the presence of proteins in the solution. The concentration of peptide bonds in the solution contributes to the intensity of the purple colour. Short-chain peptides often yield pink or blue colours in the biuret test.

Requirements

  • Protein solution- bovine serum albumin (BSA)/ egg albumin.
  • Biuret reagent:

    • Sodium hydroxide (NaOH) or potassium hydroxide (KOH) (10% w/v)- 300ml 
    • Copper (II) sulfate pentahydrate – 1.5 gm
    • Potassium sodium tartrate (5H2O) (also known as alum or Rochelle salt) – 6.0 gm
    • Potassium iodide (optional)- 1.0 gm
    • Distilled water – 1000 ml (total volume)
  • Water bath (optional). (To maintain the temperature).
  • 3 clean & dry test tubes.
  • Pipette

Cu2+ ions from CuSO4 are important components as they react with the peptide bonds to form coordinate complexes.

NaOH provides an alkaline environment, which helps dissolve the proteins as the experiment can only work in the liquid form. It is taken in a 10% weight to volume ratio.

Rochelle salt (known as ‘fitkari’ in Hindi) acts as the chelating agent & stabilizes the Copper (II) ions.

Potassium iodide inhibits the reduction of copper from Copper (II) to copper (I). 

The Biuret reagent is a solution made of hydrated Copper (II) sulfate and Sodium hydroxide or Potassium hydroxide. Sodium hydroxide impacts the alkaline medium, and potassium sodium tartrate (alum) is added to chelate hence stabilizing the cupric (Copper(II)) ions in the solution or maintaining their solubility in alkaline solution.

If a water bath is not available, you can perform the experiment at room temperature.

Procedure

  1. Take 3 clean & dry test tubes.
  2. Add 1-2 ml of test solution, egg albumin, distilled water in the respective test tubes.
  3. Add the same amount, i.e., 1-2 ml of biuret reagent, in each test tube.
  4. Shake well & allow the mixture to rest for 5 minutes.
  5. Observe the colour changes.

Observation

  1. Negative biuret test- no change in colour, i.e., the solution remains blue, which signifies the absence of protein in the solution.
  2. Positive biuret test- the solution turns from blue to violet/ deep purple, confirming the presence of proteins in the solution.

Conclusion

  • A negative biuret test means the absence of protein in the solution & a positive biuret test means there are proteins/ peptide bonds present in the solution.
  • The intensity of colour depends upon the quantity of the peptides. The higher the number of peptide bonds, the deeper the violet colour will be.

Advantages

  • It has a short turnaround time with only a few interfering substances.

Disadvantages

  • Low sensitivity.
  • Interference by ammonium sulfate in colour development.
  • At least two peptide bonds are needed.

Alternatives

The two sensitive alternatives to the biuret test are the Lowry assay and the bicinchoninic acid (BCA) assay. In these tests, the copper (I) (Cu ) formed during the biuret reaction reacts further with other reagents, leading to a deeper colour.

Copper (I) Cu forms a deep purple complex with bicinchoninic acid (BCA), which absorbs the light around 562 nm, producing the trademark mauve colour in the BCA test. The water-soluble BCA/copper complex is much stronger in absorption than the peptide/copper complex, thus increasing the sensitivity of the biuret test by a factor of around 100. The BCA assay permitted the detection of proteins in (0.0005 to 2 mg/mL). In Addition, the BCA protein assay provides the advantage of compatibility with substances.

In the Lowry protein assay, Folin–Ciocalteu reagent is used, which oxidizes copper (I) (Cu ) back to Cu2 by MoVI present in it, which forms molybdenum blue (MoIV). Under these circumstances, Tyrosine residues in the protein also form molybdenum blue. This allows proteins to be detected in concentrations between 0.005 and 2 mg/mL. Molybdenum blue, in turn, can bind certain organic dyes such as Auramine O and malachite green, resulting in further amplification of the signal.

Points to note:

  • Biuret test is also known as the Piotrowski test, in the name of the scientist who first documented it. It is a chemical test used to detect the presence of peptide bonds in a solution, used to give qualitative & quantitative estimations of protein. Protein in the range of 5–160 mg/mL can be determined.
  • Proteins are amino acid polymers held together by peptide bonds.
  • Peptide bonds are formed between two amino acids when the carboxyl group of one molecule of an amino acid reacts with an amino group of another amino acid molecule. Water is produced as the by-product of this reaction. It is a type of condensation dehydration reaction.
  • No alpha-amino acids other than histidine give a positive result on their own. To get a positive test, there must be at least two amino acids joined together by the peptide bond.
  • Biuret is not used for the experiment. It’s just named so because the peptides react the same way as the biurets.
  • For this experiment to give the result, there must be at least two peptide bonds, i.e., at least three amino acids.

Conclusion

These were the fundamentals regarding experimenting using the biuret test on urea. You can use the same or similar methods to test protein presence in other compounds. You can also adopt any of the given alternatives to do the same.

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