Introduction Proteins are organic compound that are arranged in a linear chain. The word ‘ protein’ refers to a type of molecule in food that can be broken down into amino acids. The body needs twenty amino acids to function as a biological machine and it can synthesize eleven of these by itself. However there are nine, called ‘essential amino acids’ that the body cannot create and has to gain through the consumption of food. When we eat, the body breaks down the protein in food in order to create the amino acids that it needs. Proteins are large biomolecules, or macromolecules, consisting of one or more long c hains of amino acid residues. Proteins perform a vast array of functions within living organisms, including catalyzing metabolic reactions, DNA replication, responding to stimuli, and transporting molecules from one location to another. Proteins differ from one another primarily in their sequence of amino acids, which is dictated by the nucleotide sequence of their ge nes, and which usually results in protein folding into a specific three-dimensional structure that determines its activity. (Wikipedia) Amino acids are biologically important organic compounds containing amine (-NH2) and carboxylic acid (-COOH) functional groups, usually along with a side-chain specific to each amino acid. The properties o f the side chain are what determines the classification of the amino acid into weak acid, weak base, hydrophilic and hydrophobic. (Wikipedia) The main objective of this experiment is to study the general reactions re actions of proteins and to study the R groups of the proteins. These objectives are achieved by carrying out several tests such as Biuret test, Ninhydrin test, Xanthoproteic Test, Millon’s test and Sakaguchi’s test. (Biochem practical Manual)
Results Biuret Test No 1
Sample Bovine serum albumin
Observation The solution turns from blue to dark purple/violet
2
Casein
The solution turns from blue to light purple/violet
3
Glycine
No changes. The solution remains in blue colour
4
Urea
No changes. The solution remains in blue colour.
5
H20(water)
No changes. The solution remains in blue colour.
Interpretation Protein is present. It confirms the presence of peptide bonds in the protein Protein is present. It confirms the presence of peptide bonds in the protein Protein is absent . Glycine is an amino acid. It confirms the absence of peptide bonds Protein is absent. Urea is result of the breakdown of amino acid. Hence the peptide bonds are absent Protein is absent. Water acts as a negative control of the experiment. Peptide bonds are absent.
Ninhydrin Test No 1
Sample Glycine
Observation A violet blue solution is formed
2
Proline
Dark yellow colour solution with a white precipitate is formed.
3
Bovine serum albumin
A clear solution is formed
Interpretation Free amino acid group is present in glycine. Proline is an imino acid. It is a secondary amino acid and it has a ring structure. Hence the free amino acid group is absent.
Xanthoproteic Test No 1
Sample Glycine
Observation Colourless solution is formed
2
Phenylalanine
Pale yellow solution is formed
3
Bovine serum albumin
Orange red solution is formed
Interpretation The benzene ring is absent. Since glycine is aliphatic amino acid it do not possess a benzene ring. Phenylalanine is an aromatic amino acid. It has a benzene structure . This confirms thatbovine serum albumin is an amino acid containing a benzene ring
Millon’s test
No 1
Sample Glycine
2 3
Bovine serum albumin Casein
Observation No changes are observed. Clear solution is formed Dark reddish pink precipitate is formed Light reddish pink precipitate is formed
Interpretation Protein is absent since glycine is an amino acid Protein is present Protein is present
Sakaguchi’s test
No 1
Sample Glycine
2
Arginine
3
Urea
Observation Light yellow solution is formed Light reddish pink solution is formed Light yellow solution is formed
Interpretation Arginine(a type of amino acid) is absent Arginine is present Arginine is absent
Discussion Proteins are organic compounds made of amino acids.The amino acids which is the building block of proteins joined together by the peptide bonds. In a normal functional protein, the long chain of amino acids is folded into a 3- dimensional shape. Every protein has a unique 3-dimensional shape that is suited to its biological function in a living organism. The unfolding of the 3D shape will denature the protein where the protein will no longer be able to carry out its biological function and will usually be destroyed by the cell.
There are several quantitative tests for determining whether amino acids or proteins are present in solution. These tests are specific for the presence of peptide bonds,certain types of side chains and the type of secondary structure present. The biuret test is a chemical test used for detecting the presence of peptide bonds. In the presence of peptides, a copper(II) ion forms violet-colored coordination complexes in an alkaline solution. The Biuret reagent contains copper ions which give it a blue color. The copper ions will interact with a compound that contains two or more peptide bonds, resulting in the formation of a violet/purple-colored product. The dipeptide bonds in proteins react with the Cu2+ ions to form the complex. The lone pairs pre sent on the Nitrogen of the peptide linkage bonds with the cupric ions in the reagent to create a violet purple colour change. When a compound does not have at least two peptide bonds, it will not re act with the Biuret reagent, and no purple c olor will appear . The solution will remain a shade of blue due to the copper ions. Biuret test is specific for testing Proteins, It helps to differentiate between Proteins (+ve) and Amino Acids (-ve). In the experiment above, the BSA forms the highest intensity of the purple complex compared to caseine because of the number of peptide bonds in BSA is higher than in caseine. The number of peptide bonds present in the protein is proportional to the intensity of the violet coloured complex formed. Thus, the biuret reaction is the basis for a simple and rapid colorimetric reagent of the same name for quantitatively determining total protein concentration The violet coloured complex formed confirms the presence of proteins, so in BSA and caseine proteins are present. While glycine shows negative result in the Biuret test because glycine is the simplest amino acid. A peptide bond (amide bond) is a covalent chemical bond formed between two amino acid molecules. Since there are no peptide bonds present in the glycine alone, it shows a ne gative result in the Biuret test. Urea which is the breakdown of amino acids shows a negative result in Biuret t est since the peptide bonds are absent .But when the urea is heated it may give a positive r esult because the heated urea forms the biuret compound that reacts with the cupric ions present in the reagent to form the coloured complex. Although amino acids are the building block of amino acids, but it g ive negative results in Biuret test because atleast TWO or more peptide bonds are required for the formation of the chelate complex. In single amino acids there are no peptide bonds present While in dipeptides only ONE peptide bond present hence this gives a negative result.
The biuret test can be extended to quantitatively measure the concentration of total protein using absorption spectroscopy method. Using a spectrophotometer which follows the Beer -Lambert law is the instrument which can be used in this method. Bee r Lambert law states that the absorption of the sample is directly proportional to the concentration o f the species. Hence in this case the absorption of the sample is directly proportional to the number of the peptide bonds. Ninhydrine test utilized to identify amines in particular alpha amino acids present in the solution. It is commonly used to detect the lysine in fingerprints. Ninhydrin (2,2-Dihydroxyindane-1,3-dione) is a chemical used to detect ammonia or primary and secondary amines. When reacting with these free amines, a deep blue or purple co lor known as Ruhemann's purple is produced. The ninhydrin reagent will react specifically with a primary amino functional group on a compound, resulting in the formation of a violet/purple-colored product. When a compound doesnot have a primary amino g roup, it will not react with the ninhydrin reagent, and no purple color will appear (solution will remain colorless) In the experiment above , BSA shows positive result by forming violet blue colour solution. The alpha amino group of the free amino acid react with ninhydrin to form a blue to purple colour change. However, some amino acids such as the imminoacid proline react with ninhydrin differently to form a bright- yellow colour change. This is because proline has a ring structure as shown in the diagram below.
The blueish-purple result is usually associated with primary amino acids. In these amino acids, the N is free to react with ninhydrin. However, in proline, the N is not available for reaction as it is locked in the ring structure. Therefore no ammonia is produced, so no blue color is presented. The objective of xanthoproteic test is to differentiate between aromatic amino acids which give positive results and other amino acids which do not possess the benzene ring.Amino acids containing an aromatic nucleus form yellow nitro derivatives on heating with conce ntrated HNO3. The salts of these derivatives are orange in color.
Concentrated nitric acid reacts with the aromatic rings that are derivatives of benzene giving the characteristic nitration reaction. BSA contain activated benzene rings which are easily nitrated to or ange colored compounds upon heating . The aromatic ring of phenylalanine dose not react with nitric acid despite it contains a benzene ring, but it is not activated, therefore it will not react.
Millon’s test is specific for tyrosine, the only amino acid containing a phenol group, a hydroxyl group
attached to benzene ring.The main objective of this test is to detect the presence of tyrosine in the sample. In Millon's test, the phenol group of tyrosine is first nitrated by nitric acid in the test solution.Then the nitrated tyrosine complexes mercury ions in the solution to form a brick-red solution or precipitate of nitrated tyrosine, in all cases, appearance of red color is positive test which indicates the presence of proteins. Millon’s test is given by any compound containing a phenolic hydroxy group. Consequently, any protein containing tyrosine will give a positive test of a pink to dark-red colour.However ,Millon's test is not specific for proteins (it detects phenolic compounds), and so must be confirmed by other tests for proteins such as the biuret test and the ninhydrin reaction. In the experiment above, glycine shows negative results since it is an amino acid. And it does not possess phenolic hydroxyl molecule attached to it. While BSA and caseine give positive result which is confirmed by the formation of dark red precipitate. This indicates that protein is present. Sakaguchi test is a specific qualitative test for the detection of amino acid containing gauanidium group [R-NH-C= (NH2)2+ - NH2]. In other words it’s a test for guanidines, for an example arginine. In alkaline solution, arginine react with α -naphthol and sodium hypobromite /chlorite as an oxidizing agent, to
form red complexes as a positive result.In the experiment above only in arginine thelight reddish pink solution is formed which indicates the positive result.
Conclusion In conclusion we can state that there are several experimental analysis that can be done to confirm the presence of protein and also to study the side chain, R group and structure o f the protein. Based on all the reactions above, different protein and amino acids shows different characteristic difference. Hence different tests must be done to confirm the specificity of the proteins. By the end of this experiment the objectives of this experiment were met.
