Biuret Test

The biuret test is a chemical test that can be used to see if an analyte has peptide bonds or not. As a result, the biuret test may be used to figure out how much protein is in the analyte. In this test, the presence of peptides induces the copper (II) ion to form pale purple (or mauve) coordination complexes (when the solution is sufficiently alkaline).

Overview of Biuret Test

Biuret is a compound produced by heating urea at 180 ℃. Biuret test is the name of a chemical test that utilises the Biuret reagents, which contains a 1% solution of Copper II sulphate (CuSO₄). It is the Cu₂⁺ in the Biuret reagent that forms a complex with the peptide bonds found in proteins. Hence, this test helps in determining peptide bonds in any substance.

When two acids are attached through carbonyl and amino groups, they are called peptide bonds. Moreover, the fundamental unit of protein is amino acids which are connected via peptide bonds. Notably, this experiment is a vital one and students about to appear on their boards in the near future should understand this chapter in detail.

Biuret Test Principle

This test is also known as Piotrowski’s test after the name of Gustaw Piotrowski, a polish physiologist who documented this test in 1857. Several other methods have been developed based on this method—for example, the modified Lowry test and BCA test. 

However, the mechanism of this test works through a series of principles. They are discussed below.

• In the presence of alkaline, when Biuret is reacted with dilute copper sulphate, a purple coloured substance is formed. The reason behind this colour is the formation of a chelate complex or the copper coordination complex. 

• Cu (II) or cupric ions create a chelate complex of violet colour, using oxygen of water and the unshared electron pairs of peptide nitrogen. 

• Since this complex absorbs light in 540 nm, it appears violet. In the presence of protein, it changes its colour from blue to violet. 

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• Naturally, the colour intensifies as the number of peptide bonds increases in protein.  

• Depending on this test’s principle, we can find the peptide bonds in any biological fluid. 

• This reaction takes place in a compound having at least two, H₂N-CH₂-, H₂N-C, and H₂N-CS- or similar groups attached directly or via a nitrogen or carbon atom. 

• One cupric ion is generally attached to six nearby peptide linkages through coordinate bonds.

Once you know about the principles of the Biuret test for protein, you must also concentrate on the detailed procedure of conducting. It has been discussed below in detail.

Biuret Test Manual

Materials Requirements

• 1% alanine and 5% albumin or egg white (as positive control)

• Biuret reagents

• Deionised water (as negative control)

• Dry test tubes

• Water bath

• Pipettes

Biuret Reagents

Biuret reagent is made of Copper sulphate (CuSO₄), sodium hydroxide (NaOH) and sodium-potassium tartrate (also known as Rochelle salt). Despite the name, this reagent does not contain Biuret ((H₂N-CO-)₂NH). It is a vital component of Biuret protein assay. 

Biuret Reagent Preparation

It is formed by mixing NaOH in a solution of CuSO₄, turning it alkaline. Following are the steps to yield 1000mL of Biuret reagent.

• Take distilled water (500 ml) and dissolve pentavalent copper sulphate (1.5gm) and sodium-potassium tartrate (6gm).

• Sodium potassium tartrate, a chelating substance that stabilises the copper ions.

• Now take 2 molar hydroxide (375 ml)

• Now take a volumetric flask and mix two solutions.

• Finally, make it to 1000 ml by pouring distilled water.

Procedure

Using the following steps, you can easily conduct this test.

• First, take 3 dry and clean test tubes.

• Now add 1 or 2 mL of the test solution, albumin and deionised water in the test tubes.

• Add Biuret reagent (1-2 mL) in each test tube. 

• Now shake the solution well and let it stand for 5 minutes. 

• Finally, observe how the colour changes. 

Precaution

Often ammonium and magnesium ions hinder this test. However, using excess alkali, it can be removed. 

Observation

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Following is the Observation and Analysis of the Result for Each Sample Solution

Positive Result

• The Colour turns purple.

• All proteins and peptides give positive results. 

• Only amino acid, Histidine, gives a positive result.

Negative Result

• No change in colour.

• Also, to ensure that the test sample is alkaline, add a few drops of 5% sodium hydroxide solution to each test tube. Moreover, if you are using a Biuret reagent, ensure that you shake that well as it is prepared with a reagent of 1% Copper II sulphate and 5% sodium hydroxide.

Note: Do not forget to show your understanding of why 5% sodium hydroxide alkalises the sample and 1% Copper II sulphate forms a complex with peptide bonds with chemical reactions.

Increasing Biuret Test Sensitivity

Cu+ is a strong reducing agent that can react with Mo(VI) during Folin-Ciocalteu's test to produce molybdenum blue. Following this way, primarily proteins are detected in the concentrations between 0.005 and 2 mg/mL. On the other hand, Molybdenum blue can bind a few specific organic dyes such as malachite green and Auramine o. It results in the further amplification of the signal.

Cu⁺ ions produce a deep purple complex with BCA or bicinchoninic acid. It allows detection of the proteins with a range of 0.0005 to 2 mg/mL. This is also referred to as "Pierce assay", honouring a reagent kit manufacturer.

Advantages 

• This method is the simplest and a rapid way to detect protein in a sample. Also, it is less expensive than the Kjeldahl test. 

• It provides a stable colour; hence, it does not cause deviations like other methods like UV absorption, Folin-Lowry, etc. 

• Apart from protein, very few compounds interfere in the test. 

• It only identifies N from protein or peptide bonds. It means it does not detect non-protein nitrogen.

Disadvantages

• It is not as sensitive as the Folin Lowry test. Nonetheless, it is necessary for at least 2-4 mg protein to be detected. 

• A high concentration of ammonium salts and bile pigments influence the reaction. 

• It shows different colours for different proteins. For example, gelatine provides a pink-purple colour. Also, carbohydrates and lipids take away the clarity of the given solution.

• The proteins have to be soluble for detection.

• It is not an absolute test. The colours need to be standardised for known proteins such as BSA.

• You can hasten the test simply by heating or by adding 30% isopropyl alcohol. It can reduce the reaction time from 35 to 10.

Significance of Biuret Test

• It is widely used in determining the protein amount in urine. 

• Biuret test for protein is also useful to total protein's quantitative determination, using spectrophotometric analysis.

Biuret Test for Various Substances

The casein and pure protein content of skimmed milk, as well as whole milk's protein content, can be measured with the help of Biuret test solutions which consist of potassium hydroxide and a detergent. The Biuret test for protein is useful in the case of both cheese and meat as they get dissolved in potash lye or solutions of alkaline detergent. However, fat, lactose or turbidity disturbs this test. By extraction or additional measurements with a zinc-containing, copper-free, Biuret reagent, they are removed after mixing hydrogen peroxide.

Test Your Knowledge

1. Which colour do we get for a negative Biuret test?

1. Red

2. Black

3. Blue

4. Orange

Answer: (c)

2. What makes the Biuret reagent purple?

1. Peptide bonds

2. Amide bond

3. Hydrogen bond

4. Glycosidic bond

Answer: (a). 

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