Tag: Biuret Test For Protein
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Biuret Test for Protein: Principle, Procedure, and...
Continue ReadingBiuret 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.
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Biuret Test For Protein: Definition, Principle, and...
Continue ReadingWhat is Biuret Test?
Biuret is a compound which is produced by heating the urea at a temperature of 180ºC. Biuret test is one of the chemical tests which uses Biuret reagents that contains 1% of copper II sulphate.
Cu2+ which is present in the Biuret agent and forms a complex with the peptide bond in the proteins. Thus, this test helps in determining the peptide bonds in the substance.
When two acids are attached through amino and carbonyl group, they are called as peptide bonds.
The fundamental unit of protein is usually termed as amino acids that are connected through peptide bonds.
Biuret Test Principle
Biuret test is commonly called as Piotrowski’s test, which is named after the physiologist Gustaw Piotrowski, who first documented this test in the year 1857.
Other new methods have been developed based on the principle used in this method such as Modified Lowry test and the BCA tests.
Biuret test works with the certain set of principles and they are listed below. When Biuret reacts with dilute copper sulphate in a presence of alkaline, a purple-colored substance is formed, this color is formed due to the chelate complex or due to the copper co ordination complex.
Cupric ions chelate a violet-colored complex using the oxygen of water and the unshared electron pairs of the peptide nitrogen.
As this complex absorbs light in 540nm, the sample turns into a purple color at the present of protein complexes. And hence it changes color from blue to violet.
However, color intensifies depending on the number of peptide bonds according to the increase in number of protein molecules.
Depending on the above principle, we can detect the amount of peptide bonds that were present in any of the biological fluids.
This reaction occurs in a compound which have at least 2, H2N-CH2-, H2N-C, and H2N-CS groups or other similar groups which are directly attached to the nitrogen or a carbon atom.
Generally, one molecule of cupric ion is attached to the six peptide linkages through the coordinate bonds.
Materials and Reagents Required for Biuret Test
Materials:
 1% of alanine and 5% of egg white or albumin for the positive control
 Deionized water, as negative control
 Biuret reagents
 Dry test tubes
 Water bath
 Pipettes
Reagents:
Biuret reagent is usually made up of copper sulphate, Sodium hydroxide and sodium tartrate, which is also known as Rochelle salt. It also contains a reagent named Biuret which is very important for this Biuret protein assay.
Preparation of Biuret Reagent
Biuret reagent is usually formed by mixing sodium hydroxide with a solution of copper sulphate which turns it into alkaline. Then the biuret reagent is yield as 1000 ml by the following steps.
 First, 500 ml of distilled water is taken and it is dissolved with pentavalent copper sulphate of 1.5grams and further with sodium potassium tartrate of 6 grams.
 Sodium potassium tartrate contains chelating substance that helps in stabilising the ions of copper.
 Then 375ml of molar hydroxide is taken and both the solutions are mixed in the volumetric flask.
 Hence finally 1000ml is made by pouring a distilled water into the flask.
Biuret Test Procedure
 Initially 3 dry test tubes are taken.
 1 to 2ml of test solution, albumin and the deionised water are added into the test tubes.
 Then 2 ml of Biuret reagent is added in each of the test tubes.
 The test tubes are shaken well and it is left undisturbed for 5 minutes.
 And finally, the colour changes are observed.
Biuret Test Observation and Interpretations
Observation Interpretation No change in colour Absence of proteins Solution turns from blue to violet Presence of proteins Solution turns from blue to pink Presence of proteins Biuret Test Result
 In positive controls the colour changes from blue to purple and thus indicates the presence of proteins. However only amino acids and Histidine gives a positive result.
 In negative controls there will be no colour change.
Significance of Biuret Test
Protein is usually a complex substance which are formed by thousands of amino acids.
Amino acids are considered as amphoteric electrolytes having a group of carboxyl and amino and acts like an acid and base.
Amino acid consists of one positive and one negative charge and hence these ions are said to be electrically neutral and it does not migrate in the electrical field.
The two amino acid are linked together with a help of peptide bond which yields dipeptide.
The bond is formed between alpha-carboxy group of another amino acid and the alpha-carboxyl group oof the other amino acid by the removal of water molecule.
Peptide bond is formed as the result of condensation reaction. At the same time, three amino acids are linked together but two peptide bonds and are called as tripeptide.
During the formation of tripeptide, the chain elongates and it forms a polypeptide.
Generally, Biuret is a complex compound which is formed when heating urea at 180ºC and results in a condensation of two molecules of urea.
The peptide bond in a Biuret gives a positive result of the test. Biuret test is considered as a generalized test for detecting the proteins and peptides which has more than one peptide bond.
Biuret Test Citations
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