Biuret Test for Protein: Principle, Procedure, and Result
Biuret Test Introduction
Many of a biochemical test are used to detect the ability of the microorganism to utilize the enzymes and the proteins in the medium. Biuret test is one such test, which is used to detect the proteins in a compound.
What is Biuret Test?
Protein are one of the complex molecules which are formed by millions of amino acids.
Amino acids are known as amphoteric electrolytes as they have carboxyl and the amino groups and it acts like an acid and base, it has one positive charge and one negative charge and these ions are considered as electrically neutral and they do not migrate in the electrical field.
The two amino acids are linked together with the help of a peptide bond which results in the formation of a dipeptide and this process is often called as condensation reaction.
However, the amino acids are linked together with the help of three peptide bonds and they are known as tripeptide and as well as the chain elongates, it is also called as polypeptide.
Biuret is one of the compounds which is formed by heating urea at 180ºC, which results in condensation with two molecules of urea.
The peptide bond in the Biuret usually gives a positive result in the tests.
Biuret test is considered as one of the generally performed tests for the compounds such as proteins, which have their two or more peptide bonds.
Biuret Test Objective
 The main aim of the test is to detect the protein in the given sample or a solution.
 To detect the presence of the peptide pond.
Biuret Test Principle
As mentioned above, biuret test is one of the biochemical tests, which is used to detect the presence of a peptide bond in the compounds or substances given.
This test is purely based on the structure of peptide which consists of about two peptide linkages and results in producing a violet or purple color when it is treated along with the copper sulfate.
During the presence of an alkaline solution, the blue colored copper II ion, forms a complex with the peptide bonds.
These peptide bonds does not share their pair of electrons with the nitrogen and oxygen that is present in the water.
The colored coordination complexes is usually formed between the ion carbonyl oxygen and the amide nitrogen (=NH) of the peptide bond.
On formation, of this complex the solution changes its color blue to purple. When the purple color changes deeper, in the number of peptide copper complexes.
This compound containing at least two H2N-C, H2N-CH2-, H2N-CS- or similar groups they are joined together either directly or with the help of a carbon or a nitrogen atom.
One copper ion is appropriately linked to about 6 peptide bonds present in the molecule of protein which reacts and also the number of a protein molecules present in reaction system.
The Biuret reagent is a solution composed of Sodium hydroxide or potassium hydroxide along with hydrated copper II sulfate, and potassium sodium tartrate.
Sodium hydroxide and potassium hydroxide thus provides the alkaline medium and the potassium sodium tartrate is added to chelate and to stabilize the cupric ions in the solution or in order to maintain their solubility in alkaline solution.
Biuret Test Reagents
 5% of egg white (albumin)
 Biuret reagent
 Water bath
 Pipettes
 Dry test tubes
Biuret Reagents:
 Copper sulfate
 Sodium hydroxide
 Sodium potassium tartarate (Rochelle Salt)
Preparation of Biuret Reagents
 This reagent is prepared by adding sodium hydroxide and copper sulfate solution, making it alkaline.
 To prepare 1000 ml of a Biuret reagent, about 1.5 gram of a pentavalent copper sulphate and about 6 gram of sodium potassium tartarate and dissolve in a 500ml of distilled water.
 Sodium potassium acts as a chelating agent and it also helps stabilize the copper ion.
 Further 375 ml of a two molar sodium hydroxide and it is mixed both the solution in volumetric flask and make it final volume to 1000 ml by adding water.
Biuret Test Procedure
 About 1ml of test solutions are taken in a dry test tubes and in another tube take 1 ml distilled water is added.
 To this about 1ml of biuret reagent to all the test tubes and it is mixed well.
 Then the test tube is observed for the developed for blue colors.
 Then there will be change in color to the purple.
 Hence, it is concluded that there is a presence of peptides and proteins and it resulted in positive tests.
 It is also to be noted that in a biuret test of protein, Histidine is the only amino acid to give a positive result.
Biuret Test Result
| Observation | Interpretation |
| If there is no color change and the solution remains blue | Absence of proteins or peptides Here the test is detected as negative |
| The solution changes into deep purple | It determines the presence of proteins. Here the test is detected as positive. |
Biuret Test Uses
 This test is generally used to detect the amount of protein present in the urine.
 Biuret reaction with the protein is applied to determine quantitative analysis of the total protein by using the spectrophotometric analysis.
Biuret Test Citations
- The sensitivity of approved Ninhydrin and Biuret tests in the assessment of protein contamination on surgical steel as an aid to prevent iatrogenic prion transmission. J Hosp Infect . 2006 Nov;64(3):288-92.
- Comparison of tests for failure of passive transfer in neonatal calf serum using total protein refractometry and the biuret method. Prev Vet Med . 2021 Apr;189:105290.Â
- Comparison of refractometry and biuret assay for measurement of total protein concentration in canine abdominal and pleural fluid specimens. J Am Vet Med Assoc . 2016 Apr 1;248(7):789-94.
Share