Biuret is a compound produced by heating urea at 180 ℃. Biuret test is the name of a chemical test which 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 group, 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 their boards in the near future should understand this chapter in detail.
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 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 Biuret test for protein, you must also concentrate on the detailed procedure of conducting. It has been discussed below in detail.
1% alanine and 5% albumin or egg white (as positive control)
Deionised water (as negative control)
Dry test tubes
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.
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.
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.
Often ammonium and magnesium ions hinder this test. However, using excess alkali, it can be removed.
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Colour turns purple.
All proteins and peptides give positive.
Only amino acid, Histidine, gives a positive 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 Biuret reagent ensure that you shake that well as it is a prepared with 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.
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 detecting 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.
This method is the simplest and a rapid way to detect protein in a sample. Also, it is less expensive than the Kjedahl 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.
It is not as sensitive as Folin Lowry test. Nonetheless, it 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.
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.
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.
1. Which colour do we get for a negative Biuret test?
2. What makes the Biuret reagent purple?
Answers: 1-c), 2-a).
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1. Which Free Amino Acids Give Positive Biuret Test?
Ans. Histidine is the only amino acid that gives a positive result to the Biuret test. The reason behind it is that, in the absence of additional reducing agents, it acts itself as a reducing agent. However, if you add Histidine in large quantity, the reaction may alter to some degree.
2. What is the Use of CuSO₄ in Biuret Test for Protein?
Ans. CuSO₄ is used in Biuret test because it is the main source of cupric II ions (Cu++) in the solution.
3. Why Biuret Solution is Important?
Ans. Biuret solution is important primarily to test the presence of protein in any substance. Apart from that, it is also used to quantify the protein content in urine. As a matter of fact, presence of excess protein in urine can result in kidney diseases and other complications like high pressure, diabetes mellitus, etc.
4. How Does Colour Change in Biuret Test?
Ans. The Biuret test is useful in finding out the protein in any substance mainly through the changing of colours. For example, in the presence of protein, it turns pink-purple.