Read this article to learn about the qualitative and quantitative tests for amino acids and proteins.
There are six tests for the detection of functional groups in amino acids and proteins. The six tests are: (1) Ninhydrin Test (2) Biuret Test (3) Xanthoproteic Test (4) Millon’s Test (5) Hopkins-Cole Test and (6) Nitroprusside Test.
We divide the food we consume into three main classes: carbohydrates, the body’s most readily available energy source; lipids, the body’s principal energy reserve; and proteins, the body’s source of energy for growth and cellular maintenance.
Proteins also make up the second largest portion of cells, after water. They are large polymeric compounds that cells synthesize from various building blocks called amino acids.
The general structure for an amino acid is shown in the following figure:
Note that all amino acids contain carboxylic acid groups (—COOH), amino groups (—NH2), and substituent or replaceable side chains (—R). Twenty different amino acids, which differ only in the structures of their side chains, are used by human cells to build proteins. The side chain structure determines the class of the amino acid: nonpolar, neutral, basic, or acidic.
Human cells can synthesize most of the amino acids they need to build proteins. However, about 8 amino acids called essential amino acids cannot be synthesized by human cells and must be obtained from food. Amino acids incorporated into proteins are covalently linked by peptide bonds. Peptide bonds are amide bonds formed between the carboxylic acid group of one amino acid and the amino group of a second amino acid.
Equation 1 below shows a peptide linkage formation between two amino acids:
The peptide bond is indicated. Note that, because every amino acid contains at least one amino group and one carboxylic acid group, it is possible for a peptide bond to form in two different ways. For example, with glycine and valine, it is also possible for the peptide bond to form between the carboxylic acid group of valine and the amino group of glycine, producing valylglycine.
Proteins are composed of hundreds of amino acids linked by peptide bonds, forming a peptide chain. We define the direction in which the amino acids link by referring to the two ends of the chain as the N-terminus and the C-terminus. The N-terminus is the terminal amino acid in the chain that contains the only amino group not part of a peptide bond.
The C-terminus is the other terminal amino acid in the chain, containing the only carboxylic acid group not part of a peptide bond. Note that the N-terminus and the C-terminus are not determined by the side chains. The number of constituent amino acids and the order, in which they are linked starting from the N-terminus, are referred to as the protein’s primary structure.
I. Amino Acid and Protein Solubility:
The physical properties of amino acids and proteins are mainly a result of their structure, both in the solid state and in various solutions. In this part of the experiment you will investigate the solubility of selected amino acids and proteins in various solutions. Using your data you will compare amino acid and protein structural characteristics.
Solubility as a Function of Solution pH:
The presence of amino and carboxylic acid groups enables amino acids to accept protons from and donate protons to aqueous solution, and, therefore, to act as acids and bases. Because proteins contain both acidic and basic side-chains, they too can donate or accept protons. A molecule that functions simultaneously as an acid and a base is known as an amphoteric molecule.
In neutral aqueous solutions, amino acids act as amphoteric molecules. For example, an amino acid with a neutral side chain contains two charges: one positive, due to the protonation of the amino group, and one negative, due to the dissociation of the carboxylic acid proton. This double ionic form of an amino acid is the zwitterionic form. Following figure shows an amino acid in the zwitterionic form.
Amino acids in zwitterionic form have many physical properties that are also associated with ionic salts. For example, zwitterionic amino acids are colourless, nonvolatile, crystalline solids with melting points above 200°C, usually melting with decomposition. They are soluble in water but not in nonpolar organic solvents such as cyclohexane.
Compared to organic amines and carboxylic acids of similar molecular weight, amino acids have much lower melting points and are highly soluble in polar organic solvents, but only slightly soluble in water. The amino and carboxylic acid groups of constituent amino acids, as well as the nature of various side-chains, allow proteins to possess some of these same properties. However, there are many other factors that must be considered when discussing protein solubility.
The solubility of amino acids and proteins is largely dependent on the solution pH. The structural changes in an amino acid or protein that take place at different pH values alter the relative solubility of the molecule. In acidic solutions, both amino and carboxylic groups are protonated. In basic solutions, both groups are un-protonated. Following figure shows an amino acid in acidic, neutral, and basic solutions.
The pH value at which the concentrations of anionic and cationic groups are equal is the isoelectric point for that amino acid or protein. Amino acids and proteins are least soluble at their isoelectric points. Most of the proteins found in human tissues and fluids have isoelectric points below pH 7.0 (below human body pH) and, therefore, exist mostly in their anionic forms.
II. Chemical Reactions of Amino Acid and Protein Functional Groups:
Certain functional groups in amino acids and proteins can react to produce characteristically coloured products. The colour intensity of the product formed by a particular group varies among proteins in proportion to the number of reacting functional or free groups present and their accessibility to the reagent. Now we will discuss various colour-producing reagents (dyes) to qualitatively detect the presence of certain functional groups in amino acids and proteins.
Amino acids contain a free amino group and a free carboxylic acid group that react together with ninhydrin to produce a coloured product. When an amino group is attached to the first, or alpha, carbon on the amino acid’s carbon chain, the amino group’s nitrogen atom is part of a blue-purple product, as shown in Equation 2. Proteins also contain free amino groups on the alpha carbon and can react with ninhydrin to produce a blue-purple product.
Amino acids that have secondary amino group attachments also react with ninhydrin. However, when the amino group is secondary, the condensation product is yellow. For example, the amino acid proline, which contains a secondary amino group, reacts with ninhydrin, as shown in Equation 3. Blue-purple and yellow reaction products positively identify free amino groups on amino acids and proteins.
The biuret test for proteins positively identifies the presence of proteins in solution with a deep violet colour. Biuret, H2NCONHCONH2, reacts with copper (II) ions in a basic solution to form a deep violet complex. The peptide linkages in proteins resemble those in biuret and also form deep violet complexes with basic copper (II) ions in solution. The general or biuret complex formed between the protein linkages and the copper (II) ion of the biuret test is shown in following figure.
Some amino acids contain aromatic groups that are derivatives of benzene. These aromatic groups can undergo reactions that are characteristic of benzene and benzene derivatives. One such reaction is the nitration of a benzene ring with nitric acid. The amino acids tyrosine and tryptophan contain activated benzene rings and readily undergo nitration.
The amino acid phenylalanine also contains a benzene ring, but the ring is not activated and, therefore, does not readily undergo nitration. This nitration reaction, when used to identify the presence of an activated benzene ring, is commonly known as the xanthoproteic test, because the product is yellow.
Xanthoproteic comes from the Greek word xanthos, which means yellow. The intensity of the yellow colour deepens when the reaction occurs in basic solution. This reaction is one of the reactions that occur if you spill a concentrated solution of nitric acid onto your skin. The proteins in skin contain tyrosine and tryptophan, which become nitrated and turn yellow.
Millon’s test is a test specific for tyrosine, the only amino acid containing a phenol group, a hydroxyl group attached to a benzene ring. 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 (I) and mercury (II) ions in the solution to form a red precipitate or a red solution, both positive results. Proteins that contain tyrosine will, therefore, yield a positive result.
However, some proteins containing tyrosine initially forms a white precipitate that turns red when heated, while others form a red solution immediately. Both results are considered positive. Note that any compound with a phenol group will yield a positive test, so one should be certain that the sample that is to be tested does not contain any phenols other than those present in tyrosine.
The Hopkins-Cole test is specific for tryptophan, the only amino acid containing an indole group. The indole ring reacts with glyoxylic acid in the presence of a strong acid to form a violet cyclic product. The Hopkins-Cole reagent only reacts with proteins containing tryptophan. The protein solution is hydrolyzed by the concentrated sulphuric acid at the solution interface. Once the tryptophan is free, it reacts with the glyoxylic acid to form the violet product.
The nitroprusside test is specific for cysteine, the only amino acid containing a sulfhydryl group (—SH). The group reacts with nitroprusside in alkaline solution to yield a red complex.