Amino acids are monomers for proteins. Protein molecules are composed of a large number of amino acids joined together by peptide bonds. There are 20 naturally occurring amino acids each of which contains an amino group (-NH2), a carboxyl group (-COOH), a hydrogen (-H) and a variable group (designated as -R). A peptide bond is formed when the carboxyl group of one amino acid bonds with the amino group of other amino acid to form a primary protein structure. This chain of amino acids, joined by peptide bonds, forms a secondary protein structure by coiling into an alpha (a) helix or into a beta (b) pleated sheets. A tertiary protein structure results when this alpha helix or beta pleated sheet folds back on itself. Two or more proteins join together to form a quaternary protein structure.
Biuret test is basically a test for the peptide bonds that link individual amino acids into a protein chain. Biuret reagent is a two part system. Dilute copper sulphate (CuSO4) forms the first part and a strong alkaline solution of potassium hydroxide (KOH) as the second part. KOH is used for denaturing the protein and breaking its hydrogen or other weak covalent bonds. If peptides or proteins are present, Cu+2 ions from the Biuret reagent combine with the peptide bonds. The resulting solution acquires a violet color whose tone depends on the amount of protein present. If the peptides are absent in the test solution, then the solution retains the blue color of the Cu+2 ions in the reagent.
In the testing conditions, certain controls are also employed. A known chemical, which does not contain the testes substance (protein in this case), is used as the negative control. Distilled water is used as negative control in the Biuret test. Another known chemical, which surely contains the tested substance (i.e. protein in this test), is used as positive control. Serum albumin can be used as a positive control for the testing of peptides. The color change between the positive and negative control solutions is used for comparison with substance whose composition is unknown.
MATERIALS REQUIRED- test tubes, pipette, control solutions- distilled water and serum albumin solution, dilute CuSO4 solution, KOH solution and the test solution.
· Prepare the control solution.
· In three different test tubes, take the test solution, the negative control- distilled water and the positive control- the albumin solution.
· Note the original colors of each of the three solutions.
· In each test tube, add about 5 drops of dilute CuSO4 solution and tap the side of the test tube slightly with the finger to mix the contents.
· After about 15- 20 seconds, add about 10 drops of KOH solution to each test tube and tap slightly to mix.
· Record the colors of each solution.
RESULT: – The negative control distilled water will retain the light blue color of the dilute CuSO4 solution. The positive control solution of albumin will show a very deep violet color. The unknown solution will be a blue if protein/ peptide are absent or will show a violet color if peptides are present. The violet color will be dark or light depending on the concentration of the protein in the solution.
An ideal observation table may have the following form: –
The color of Biuret Reagent is:
Final color after 2 minutes at room temperature
Is this a positive test? Are peptide bonds present (Yes or no?)
Negative control: water
Positive control: serum albumin
Do these foods contain proteins? (test solutions)
mashed up potato
mashed up beans
DISCUSSION:- We use distilled or de- ionized water as the negative control as we know for sure the absence of protein in water. So we can be sure that water will not test for protein and will retain the blue color of the CuSo4 when Biuret reagent is added to it. Albumin is used as positive control because the presence of protein in albumin needs no reiteration. So we can be sure that albumin will test positive for protein/ peptide and will turn a deep violet when Biuret reagents are added to it. the test solution, if it contains protein, will acquire a color that is midway between the blue of CuSO4 and the violet of positive control.
Some other tests also merit mention in the context of amino acid testing. Ninhydrin is a very specific and new reagent which is often used for amino acid testing in recent times. When ninhydrin reacts with amino acids, it results in the formation of a deep purple compound known as Rhueman’s Purple. The specificity of the test is a great advantage. This is so because ninhydrin does not react with all amines and thus gives reliable amino acid detection where many tests may fail because of contamination by amines. Ninhydrin reacts only with amino acids and not with all amines.
CHROMATOGRAPHY: – Chromatography is the science of separation of molecules on the basis of the differences on their structures and/pr their composition. A solution of the material to be separated is moved over a stationary support. Depending upon the structure, different molecules show different interactions with the support. Those who have strong interactions with support, move slowly and others move fast and thus can be effectively separated. There are many methods of chromatography. Some of them are discussed below.
Ion exchange chromatography: – All amino acids contain a side group (-R) which may be positively or negatively charged. If the net charge on the protein is positive, it is called a basic protein and if the net charge is negative, it is called acidic protein. In these cases, the amino acid will bind itself to a support that is oppositely charged. So based upon the charge, families of amino acids can be easily separated.
Hydrophobic interaction chromatography: – Some amino acids have hydrocarbon chains which are not charged and hence ion exchange chromatography becomes ineffective. Such hydrophobic amino acids can bind on a support which contains immobilized hydrophobic groups. The bonding is through a clustering effect and not through ionic or covalent bonds.
Gel Filtration: – This technique is based on the size and shape of the amino acid. There is no bonding between the support and the amino acid. There are holes on the support called ‘pores’ of different sizes. Smaller proteins penetrate the holes and larger molecules move around and migrate through spaces.
Affinit Chromatography: – this is the most powerful chromatography technique. It is very selective and efficient. A specific ligand is immobilized on a support so that it binds itself to the target protein. This technique can be used for any specific protein provided the ligand is available.
It can be concluded that where only a very qualitative estimation is required, Biuret test can be handy as it is less time consuming and can be performed with ease in any laboratory. But the findings are not always reliable as stated earlier. Chromatography test, on the other hand, is very specific in nature and thus can be considered reliable and efficient. But the facilities for performing the test are not available in every laboratory and the test is more costly as compared to the Biuret test.
Dickey J, 2003, Laboratory Investigations for Biology, 2nd Ed. Benjamin Cummings.
Stong, C.L. The Amateur Scientist: Various Kinds of Chromatography, Especially the Thin-Layer Method. Scientific American. March 1969 vol. 220 number 3 pages 124-128
Pellett, Peter L. and Young, Vernon R., 1980, Nutritional evaluation of Protein Foods, The United Nations University.