Qualitative tests of amino acid Amino acids are building blocks of all proteins, and are linked in series by peptide bond (-CONH-) to form the primary structure of a protein. Amino acids possess an amine group, a carboxylic acid group and a varying side chain that differs between different amino acids. There are 20 naturally occurring amino acids, which vary from one another with respect to their side chains. Their melting points are extremely high (usually exceeding 200°C), and at their pI, they exist as zwitterions, rather than as unionized molecules. Amino acids respond to all typical chemical reactions associated with compounds that contain carboxylic acid and amino groups, usually under conditions where the zwitter ions form is present in only small quantities. All amino acids (except glycine) exhibit optical activity due to the presence of an asymmetric α – Carbon atom. Amino acids with an L – configuration are present in all naturally occurring proteins, whereas those with D – forms are found in antibiotics and in bacterial cell walls.

Objectives: General information about amino acids. Qualitative tests of amino acids.

Introduction Food are divided into three classes : 1- Carbohydrate Source of energy. 2- Lipid Principal of energy reserve. 3- Proteins Energy for growth and cellular maintance.

(Building blocks of proteins which linked to peptide bond ) Amino acid structure (Building blocks of proteins which linked to peptide bond ) Each amino acid consists of : Central carbon atoms An amino acid Carboxyl group Side chain (All amino acids found in proteins have this basic structure, differing only in the structure of the R-group or the side chain.)

Classification of amino acids according source Essential amino acids: Humans incapable of forming requisite and must be Required in diet. Non essential amino acids: Not required in diet.

The simplest, and smallest, amino acid found in proteins is glycine for which the R-group is a hydrogen (H). Proline It is unique among the 20 protein-forming amino acids in that the amine nitrogen is bound to not one but two alkyl groups, thus making it a secondary amine.

Classification of amino acids according to their (polarity) in water Non-polar (Hydrophobic amino acid) : are amino acid that contain C,H in their side chain (hate water, normally buried inside the protein core) Uncharged polar. polar amino acids with a charge: amino acid that contain in their side chain O,N and they can dissolve in water ( like dissolve like ) hydrophilic (love water),tend to found on surface Basic polar (positively charged). Acidic polar (negatively charged).

At acidic pH, the carboxyl group is protonated and the amino acid is in the cationic form At neutral pH, the carboxyl group is deprotonated but the amino group is protonated. The net charge is zero; such ions are called Zwitterions At alkaline pH, the amino group is neutral –NH2 and the amino acid is in the anionic form. For acidic amino acids, the pI is given by ½(pK1 + pK2) and for basic amino acids it’s given by ½(pK2 + pK3).

Iso electric point (PI) : It is the pH value at which concentration of anionic and cationic groups are equal (i.e. the net charge of this molecule equals zero). Each amino acid have a different PI

Qualitative tests for amino acids There are number of tests to detect the presence of amino acid This is largely depend on the natural of side chain

Amino acids analysis Ninhydrin test: for α-L amino acids biuret test Xanthoproteic test: for Aromatic amino acids Lead sulfite test: detection of amino acids containing sulfhydral group (- SH) Millon's test: for amino acids containing hydroxy phenyl group Sakaguchi Test. Hopkins-Cole (Glyoxylic Acid Reaction)

Ninhydrin Principle: Ninhydrin degrades amino acids into aldehydes (on pH range 4), ammonia and CO2 through a series of reactions. The reducont product obtained from ninhydrin (hydrindantin) then reacts with NH3 and excess ninhydrin to produce an intensely blue or purple pigment, sometimes called ruhemann's purple. This reaction provides an extremely sensitive test for amino acids. alpha-amino acid + 2 ninhydrin ---> CO2 + aldehyde + final complex(BlUE) + 3H2O All amino acids that have a free amino group will give positive result (purple color) . While not free amino group-proline and hydroxy-proline (amino acids) will give a yellow color Hydrindantin is a chemical intermediate formed during the ninhydrin test for amines.

Note The amino acid proline and hydroxyproline also react with ninhydrin, but they give a yellow colored complex instead of a purple one. Besides amino acids, other complex structures such as peptides, peptones and proteins also react positively when subjected to the ninhydrin reaction. Ninhydrin_ethanol reagent is flammable. Toxic, and irritant. Keep away from Bunsen burner flames prevent eye, skin, clothing contact. Avoid inhaling the vapors or ingesting the reagent.

With all amino acid will give purple or deep blue with exception Proline gives yellow not violet (why)? The secondary amino group (imino group) of proline residues is held in rigid conformation that reduces the structural flixibility of polypeptide regions containing proline. Proline does not give ninhydrin reaction as this reagent requires free alpha amino group but proline have imino group

Procedure To 1 mL solution add 5 drops of 0.5% ninhydrine solution Boil over a water bath for 2 min. Allow to cool and observe the blue color formed. Results:

Biuret test Biuret structure: it is result of condensation of two molecule of urea Principle: The biuret reagent (copper sulfate in a strong base) reacts with peptide bonds in proteins to form a blue to violet complex known as the “biuret complex”. This color change is dependent on the number of peptide bonds in the solution, so the more protein, the more intense the change. 

The NaOH is there to raise the pH of the solution to alkaline levels; the crucial component is the copper II ion (Cu2+) from the CuSO4. When peptide bonds are present in this alkaline solution, the Cu2+ions will form a coordination complex with 4 nitrogen atoms from peptide bonds. N.B. Two peptide bonds at least are required for the formation of this complex.

A chelate is a chemical compound composed of a metal ion and a chelating agent. A chelating agent is a substance whose molecules can form several bonds to a single metal ion. In other words, a chelating agent is a multidentate ligand. . Chelation describes a particular way that ions and molecules bind metal ions. A chelate is a chemical compound composed of a metal ion and a chelating agent. A chelating agent is a substance whose molecules can form several bonds to a single metal ion. In other words, a chelating agent is a multidentate ligand.

Procedure To 2 ml of protein solution in a test tube, add 4ml of reagent incubation 30 min Result :

Interpretation Observations Proteins are not present No change ( solution remains blue ) Proteins are present The solution turns from blue to violet( purple) Peptides are present ( Peptides or peptones are short chains of amino acid residues) The solution turns from blue to pink

Xanthoproteic test Objective: to differentiate between aromatic amino acids which give positive results and other amino acids. Principle: Concentrated nitric acid react with aromatic nucleus present in the amino acid side chain [nitration reaction] giving the solution yellow color. Note: Amino acids tyrosine and tryptophan contain activated benzene rings [aromatic nucleus] which are easily nitrated to yellow colored compounds. The aromatic ring of phenyl alanine dose not react readily with nitric acid despite it contains a benzene ring, but it is not activated, therefore it will not react Yellow color Some amino acids contain aromatic groups that are derivatives of benzene. These aromatic groups can undergo reactions One such reaction is the nitration of a benzene ring with nitric acid. The amino acids that have activated benzene ring can readily undergo nitration. In the presence of activated benzene ring, forms yellow product.

Phenylalanine still reacts with nitric acid, just not as readily as Tyr or Trp because Tyr and Trp have electron donors (hydroxyl and the nitrogen heteroatom), which make the ring a lot more attractive for electrophilic nitration. To get Phe to react, you may have to boil it with nitric acid

Procedure To 2 mL amino acid solution in a boiling test tube, add equal volume of concentrated HNO3. Heat over a flame for 2 min and observe the color. Now COOL THOROUGHLY and CAUTIOSLY run in sufficient 3ml NaOH (why) Observe the color of the nitro derivativative of aromatic nucleus.

(Glyoxylic Acid Reaction) Hopkins-Cole (Glyoxylic Acid Reaction) objective: Specific for tryptophan (the only amino acid containing indole group) Principle: Reacting with a glyoxylic acid in the presence of a strong acid, the indole ring forms a violet cyclic product. The protein solution is hydrolyzed by conc. H2SO4 at the solution interface. Once the tryptophan is free, it reacts with glyoxylic acid to form violet product.

Procedure In a test tube, add to 2 ml of the solution an equal volume of Hopkins- Cole reagent and mix thoroughly. Incline the tube and let 5 to 6 ml of conc. H2S04 acid flow slowly down the side of the test tube, thus forming a reddish - violet ring at the interface of the two layers. That indicates the presence of tryptophan

Millon's test Objective: This test is specific for tyrosine. Because it is the only amino acid containing a phenol group. Note: phenol group, a hydroxyl group attached to benzene ring. Millon’s reagent contains mercury and HNO3 and is very toxic, corrosive a strong oxidant, an irritant, and can cause burns Millon's test Phenolic amino acids such as Tyrosine and its derivatives respond to this test. Compounds with a hydroxybenzene radical react with Millon’s reagent to form a red colored complex. Millon’s reagent is a solution of mercuric sulphate in sulphuric acid.

Principle: 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 , appearance of red color is positive test. Note: all phenols (compound having benzene ring and OH attached to it) give positive results in Millon’s test.

Procedure To 2 ml of protein solution in a test tube, add 3 drops of Millon’s reagent. Mix well and heat directly on a small flame. BWB 5 min A white ppt is formed with albumin and casein (but not gelatin); the ppt gradually turns into brick red.

Sakaguchi Test. Objective: detection of amino acid containing gauanidium group. In other words it’s a test for, arginine. Principle :  In alkaline solution, arginine react with α-naphthol and sodium hypobromite /chlorite as an oxidize agent, to form red complexes as a positive result.

Procedure Add 1 ml of 3 N NaOH solution to 1 ml of the protein solution, followed by addition of 0.5 ml of 0.1 % α- naphthol solution, and a few drops of 2 % sodium hypobromite solution (NaOBr). The formation of a red color indicates the presence of a guanidinium group in the compound under examination.

Lead Sulfite Test Objective: This test specific for–SH [sulfhydral group ] containing amino acid (Cysteine). Principle: - Sulfur in cystine, is converted to sodium sulfide by boiling with 10% NaOH. - The Na2S can be detected by the precipitation of PbS (lead sulfide) from an alkaline solution when adding lead acetate Pb (CH3COO)2.

Methionine and cysteine contains sulfur group Sulfur containing amino acids, such as cysteine and cystine upon boiling with sodium hydroxide (hot alkali) yield sodium sulfide. This reaction is due to partial conversion of the organic sulfur to inorganic sulfide, which can detected by precipitating it to lead sulfide, using lead acetate solution. S.(protein) + 2NaOH-------- Na2S Na2S + (CH3COO)2pb ------- PbS + 2CH3COONa

Procedure 1. Place 1 ml of 2% casein, 2% egg albumin, 2% peptone, 2% gelatine and 0.1 M cysteine into separate, labeled test tubes. 2. Add 2 ml of 10 % aqueous sodium hydroxide. Add 5 drops of 10 % lead acetate solution. 3. Stopper the tubes and shake them. Remove the stoppers and heat in a boiling water bath for 5 minutes. Cool and record the results.

That’s all Now to practical part

3ml NaOH Hopkins-Cole Ninhydrin Test Biuret test Xanthoproteic test 1 mL solution 5 drops of 0.5% ninhydrine soln Boil over a water bath for 2 min. 1 ml of protein soln 2 ml of biuret reagent incubation 30 min . 2 mL solution in a boiling test tube 2 ml of conc. HNO3. Heat over a flame for 2 min. COOL 3ml NaOH Add 2ml of soln + 2 ml of Hopkins reagent. 5-6 ml conc. H2S04 Millon’s Test Lead sulfite test Sakaguchi Test 1ml cysteine 2 ml of 10 % NaoH Add 5 drops of 10 % lead acetate soln. Put in BWB for 5 min. 2 ml of protein solution 3 drops of Millon’s reagent. Mix well and heat directly on a small flame, BWB 5 min 1 ml of the protein solution 1 ml of 3 N NaOH 0.5 ml of α- naphthol a few drops of 2 % (NaOBr).