Qualitative test of proteins

Qualitative test of proteins

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  QUALITATIVE ANALYSIS OF PROTEIN By:Neha Sheth
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  INTRODUCTION Proteins are macromoleculesand they form collodial systems. Most of them are hydrophilic, therefore, they are hydrated. Being colloids, they are charged. Proteins can be precipitated by dehydration and neutralization of the electrical charges, which carry, to bring them to the isoelectric point (isoelectric pH).
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  CLASSIFICATION: 1. Simple Proteins–made up of only amino acids. e.g.– albumins, globulins, prolamines, protamines, histones... 2. Conjugated Proteins-- amino acids+ non-Protein Part (Prosthetic grp.) e.g.—glycoproteins, lipoproteins, chromoproteins, metalloproteins, phosphoproteins… 3. Derived Proteins– derived from native proteins. Primary—formed due to some intramolecular changes not involving hydrolysis. e.g.- Proteans, metaproteins.. Secondary---formed by hydrolysis of native proteins. e.g.—proteoses and peptones.
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  TESTS FOR PROTEIN: Onthe Basis of physical and chemical properties and the presence of different amino acids, proteins in a given solution can analysed under the following heading:  Precipitation Reaction of Protein: By acid, alkali, heavy metals, alcohol, alkaloid reagent, salts, heat & isoelectric pH.  Colour Reaction of Protein: Contributed by amino acids, peptide bonds & non protein components.
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  PRECIPITATION REACTION OFPROTEIN  The stability of proteins in solution will depend mainly on the charge and hydration.  Polar groups of the proteins(-NH2,COOH,OH groups) tend to attract water molecules around them to produce a shell of hydration.  Repulsive electrostatic forces form when proteins are suspended in an electrolyte solution. This repulsive forces between proteins prevent aggregation and facilitate dissolution.
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  PROTEINS CAN BEPRECIPITATED FROM THEIR SOLUTION BY:  Addition of salt like ammonium sulphate etc..  Adjusting the pH of protein solution to its isoelectric pH.  Addition of organic solvents like alcohol etc..  Addition of alkaloidal reagents like picric acid, tricholro acetic acid, sulphosalicylic acid etc…  Addition of heavy metals like lead acetate, mercuric sulphate etc..  Heat and addition of acetic acid (or) Heat coagulation ..
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  PRECIPITATION BY SALTS: Half& Full Saturation Test:  When a neutral salt such as (NH4)2SO4 added to protein solution.  Shell of hydration remove from outer surface of protein molecules which causing protein precipitation. This is called salting out.  Amount of salt required for precipitation depends on 1) molecular weight 2) Surface area 3) Number of electric charges 4) Shell of hydration
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  TEST OBSERVATION INFERENCE 3ml of protein solution + 3 ml of saturated ammonium sulphate. Mix and let it stand for 5 minutes. Filter the contents. With the filtrate perform Biuret test as follows; 2 ml of filtrate + 2 ml of 40% NaOH + 2 drops of copper sulphate. Violet colour is observed Violet colour indicates the presence of protein in the filtrate. (Casein, Gelatin and Globulin are precipitated at half saturation with (NH4)2 SO4) 1. Precipitation by Neutral salts (ammonium sulphate): a) Half saturation test
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  TEST OBSERVATION INFERENCE 3ml of protein solution + solid ammonium sulphate with mixing till the solution is saturated (There should be some undissolved crystals at the bottom of the test tube). Let it stand for 5 minutes. Filter the contents. With the filtrate perform Biuret test as follows; 2 ml of filtrate + 2 ml of 40% NaOH + 2 drops of copper sulphate. Blue colour is observed. Blue colour indicates the absence of protein in the filtrate. Protein is Precipitated by full saturation. (Albumin precipitates at full saturation with (NH4)2 SO4) b) Full saturation test:
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  SALTING OUT Protein molecules with ashell of water molecules keeping them soluble Protein Precipitate Addition of neutral salt Water Molecules Removal of shell of hydration causing precipitation of proteins Neutral salt, (e.g. ammonium sulfate) precipitate proteins by salting out which involves the removal of the shell of hydration causing precipitation of proteins. Higher the molecular weight lesser will be salt required for the precipitation.
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  PRINCIPLE 5 ml Albumin solutionshowing larger surface area Globulin solution showing relatively lesser surface area 5 ml The molecular weight of the albumin is much less than the the globulin so albumin is not precipitated by half saturation whereas high molecular weight globulins ar. e precipitated, So that, Albumin requires only saturated solution
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  TEST OBSERVATION INFERENCE 2.Isoelectric precipitation test: 3 ml of protein + 2 drops of BCG (bromo cresol green) indicator. Blue colour indicates pH > 7, add 1% acetic acid drop wise with mixing until a green colour is obtained. pH at this stage is 4.6. Observe whether any precipitate is formed. A curdy green precipitate is observed. Proteins have minimum solubility at their isoelectric pH. Isoelectric point of casein is 4.6. When the pH of casein solution is adjusted to 4.6, casein gets precipitated 2. Isoelectric precipitation test
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  The pH atwhich the molecule carries no net charge (zwitter ion) is known as Isoelectric point (pI) The solubility and buffering capacity will be minimum and there will be no net movement of the molecule in the electrical field. And proteins gets precipitated. Isoelectric point of casein is 4.6. When the pH of casein solution is adjusted to 4.6, casein gets precipitated 1% acetic acid pH > 7 pH is 4.6 ( pI of casein ) PRINCIPLE
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  TEST OBSERVATION INFERENCE 1ml of protein + 2 ml of alcohol. Mix. White precipitate is observed Alcohol is a dehydrating agent (removes water). When alcohol is added to protein solution it reduces the amount of water needed to keep protein in solution. This results in precipitation of protein. 3. Precipitation by organic solvents (alcohol) PRINCIPLE: Organic solvents cause precipitation of proteins by the removal shell of hydration surrounding the proteins.
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  TEST OBSERVATION INFERENCE 1ml of protein + 2 ml of basic lead acetate Mix. White precipitate is observed In alkaline medium, a protein has net negative charges. Positive charge of metal ions neutralizes the negative charge on protein and forms a precipitate of lead/ mercuric proteinate. 5. Precipitation by Heavy metals (Lead acetate)
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  PRINCIPLE Proteins acquire negative chargeon adding alkali which raises the pH above the IPS of proteins. Ionic interaction between oppositely charged particles results in protein aggregation and precipitation Metal ions Proteins Protein aggregation and precipitation
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  TEST OBSERVATION INFERENCE 1ml of protein + 2 ml of sulpho salicylic acid. Mix. White Precipitate is observed In acidic medium, a protein has net positive charges. Negative charge of acid neutralizes the positive charge on protein and forms a precipitate. 4. Precipitation by Alkaloidal reagents (sulpho salicylic acid)
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  PRINCIPLE Protein Precipitate Protein acquires+ve charge in the presence of acidic medium ( pH < IEP) Anions derived from acids, e.g. Phosphotungstate, sulfosalicylate Alkaloids when dissolved lower the pH of the medium and they themselves form anions. Proteins in this acidic medium acquire positive charge and they complex with negatively charged ions in the medium. These complexes are insoluble and they are precipitated.
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  TEST OBSERVATION INFERENCE Take10 ml (half a test tube) of protein solution. Hold the tube over a flame in a slanting position & boil the upper half. The lower half serves as control. A cloudy white colour will be observed in the heated portion. Add few drops of 1% acetic acid. Cloudy white Coagulum is observed Coagulable proteins get easily denatured on heating and form coagulum. (Albumin is precipitated when denatured at its pI~5.4) 6. Heat and acetic acid test (or) Heat coagulation test
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  PRINCIPLE  Heating causedenaturation. Disruption of secondary tertiary, quaternary structures maintained by noncovalent forces cause denaturation.  Aggregation of denatured protein is referred to as coagulum. Denaturation may be reversible in some cases (not always). But coagulation is always irreverible.  Addition of acetic acid lowers the pH of the medium towards the isoelectric pH of the albumin.  At pI proteins are least soluble. So the denaturation proteins get precipitated upon adding acetic acid.
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  Heating cause denaturation. Disruption of secondarytertiary, quaternary structures Aggregation of denatured protein is referred to as coagulum coagulation is always irreverible At pI proteins are least soluble Acetic Acid
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  COLOUR REACTIONS  Proteinsreact with a variety of reagents to form colored products because of their constituent peptide bonds and amino acids.  These reactions are useful for qualitative and quantitative studies of proteins.  For the diagnosis of aminoacidurias  For the nutritional assessment  To detect the presence of proteins or amino acids in biological fluids
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  TEST OBSERVATION INFERENCE 1ml of protein solution + 2 or 3 drops of α- naphthol, mix and add 2 ml of concentrated sulphuric acid (H2SO4)along the walls of the tube carefully Reddish violet colour ring is observed at the junction of two liquids This test detects the presence of carbohydrate. Since egg albumin is a glycoprotein, this test is positive. Sulphuric acid dehydrates carbohydrate to give furfural which condenses with α- naphthol to form reddish violet coloured complex. Proteins contain Carbohydrates 1. Molisch test
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  PRINCIPLE Carbohydrate to give furfuralwhich condenses with α-naphthol to form reddish violet coloured complex This test detects the presence of carbohydrate in Protein.
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  TEST OBSERVATION INFERENCE 1ml of protein solution + 1 ml of 5%NaOH + 2 drops of copper sulphate (CuSO4) solution. Mix. Violet colour is observed This test detects the presence of peptide bonds. Thus this test is specific for proteins. A cupric ion in alkaline medium forms a violet coloured complex with peptide bond nitrogen of protein. The reaction is so named since compound Biuret (NH2CONHCONH2) also answer this tests. Minimum requirement for a positive test is the presence of two peptide bonds. (Two or more peptide linkages present. Protein present) 2. Biuret Test
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  This test detectsthe presence of peptide bonds. Thus this test is specific for proteins PRINCIPLE: The test is positive in the presence of two or more peptide bonds.
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  TEST OBSERVATION INFERENCE 1ml of protein + 10 drops of Ninhydrin. Boil strongly. Blue colour for proteins/ peptides. Yellow colour for proline and hydroxy proline Detects the presence of free amino group. Ninhydrin react with alpha- amino group of proteins and free amino acids. (Alpha Amino groups of proteins at N- terminal are responsible for positive test with proteins.) 3. Ninhydrin test
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  Ninhydrin Amino acid Ruhemann purple Detectsthe presence of free amino group. Alpha Amino groups of proteins at N- terminal are responsible for positive test with proteins PRINCIPLE React with alpha- amino group of proteins and free amino acids.
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  Hydrindantin Aldehyde
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  TEST OBSERVATION INFERENCE 3ml of protein + 1 ml of Concentrated nitric acid (HNO3). Boil for 1 min. Cool under tap water. Observe the colour. Divide the contents into 2 parts.To one, add 1 ml of 40% NaOH. Mix and observe. Yellow colour in acid medium Orange colour in alkaline medium This test detects the presence of aromatic amino acids, tyrosine and tryptophan. On heating with conc.HNO3 proteins containing aromatic amino acids form yellow colour due to the nitration of the benzene ring. The nitro compound freely ionized in alkaline medium thus intensifies the colour to orange by adding strong alkali. Nitration of phenylalanine under these conditions does not take place. ( Benzene ring present in protein. Aromatic Amino Acids Phenylalanine, Tyrosine and Tryptophan present in protein.) 4. Xanthoproteic test
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  Detects the presenceof aromatic amino acids, tyrosine and tryptophan. Phenylalanine will not give a positive response to the test even though it contain benzene ring. PRINCIPLE On heating with conc.HNO3 proteins containing aromatic amino acids form yellow colour due to the nitration of the benzene ring.
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  TEST OBSERVATION INFERENCE 1ml of protein + 1 ml of mercuric sulphate. Boil gently for 30 seconds. Add 2 drops of 1% sodium nitrite. Mix. Red colour is observed This test is given by the tyrosine amino acid which has OH group in the benzene ring. Proteins undergo mercuration and nitration in strong acidic medium to form red coloured mercury phenolate. (Hydroxyphenyl group present in protein. Tyrosine present in protein.) 5. Modified Millon’s test
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  Test is givenpositive by the tyrosine amino acid which has OH group in the benzene ring. PRINCIPLE Tyrosine has hydroxyphenyl group (Present in the core of Protein) The protein is denatured by mercuric sulphate in boiling water exposing hydroxyphenyl group. Sodium nitrite reacts with sulfuric acid to form nitrous acid. The exposed hydroxyphenyl groups react with nitrous acid and give red colour precipitates and solution. Mercury phenolate TYROSINE
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  TEST OBSERVATION INFERENCE 1ml of protein + 1 drop of 0.2% formalin + 1 drop of mercuric sulphate. Add 1 ml of concentrated sulphuric acid carefully along the sides of the test tube. Violet colour ring is observed This test is specific for tryptophan which contains the indole ring. The indole ring of tryptophan combines with aldehydes, e.g. formaldehyde in the presence of conc. sulphuric acid to form violet coloured compound. Gelatin gives a negative test as it does not contain tryptophan.( Indole group present in protein. Tryptophan present in the protein.) 6.Hopkin’s and Cole test (Aldehyde test)
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  Test is specificfor tryptophan which contains the indole ring PRINCIPLE INDOLE RING (Benzene + pyrrole) Protein solution Purple ring Conc. H2SO4 sink to the bottom The indole ring of tryptophan combines with aldehydes, e.g. formaldehyde in the presence of conc. sulphuric acid to form violet coloured compound.
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  TEST OBSERVATION INFERENCE 3ml of protein + 2 drops of 40% NaOH + 4 drops of alpha naphthol + 10 drops of bromine water. Mix. Bright red colour is observed This test is specific for Arginine which contains the guanidino group. In alkaline medium, α- napthol combines with the guanidine group of arginine to form a complex which is oxidized by sodium hypobromite to produce a caramel red colour. (Guanidino group present in protein. Arginine present in protein.) 7. Sakaguchi test
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  Test is specificfor arginine which contains the guanidino group Guanidino group In alkaline medium, α-napthol combines with the guanidine group of arginine to form a complex which is oxidized by sodium hypobromite to produce a caramel red colour. PRINCIPLE
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  TEST OBSERVATION INFERENCE 1ml of protein solution + 1 ml of 40% NaOH, boil strongly. Cool under the tap water. Now add 4 drops of Lead acetate solution and mix. Brown or black colour is observed This test is specific for Sulphur containing amino acid Cysteine and its conjugated product cys tine. On boiling with NaOH, the sulphur present in the protein is liberated as sodium sulphide. This reacts with lead acetate to form a brown or black precipitate of lead Sulphide. (Sulfhydryl group (-SH) present in protein. Cysteine & Cystine present in protein) 8. Sulphur Test
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  Test is specificfor sulphur containing amino acid cysteine and its conjugated product cystine. PRINCIPLE On boiling with NaOH, the sulphur present in the protein is liberated as Sodium sulphide. A brown or black precipitate of lead sulphide is formed
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  TEST OBSERVATION INFERENCE 5ml of casein + 1 ml of NaOH, boil strongly. Cool under the tap water and add 0.5 ml of concentrated nitric acid. Filter if necessary (to remove any undigested casein). Add a pinch of solid ammonium molybdate and warm gently. Canary yellow colour is observed This test is specific for organic phosphate (as casein is a phosphorprotein) which is converted to yellow ammonium phosphor molybdate. 9. Modified Neuman’s test [Test for organic phosphorus]
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  This test isspecific for organic phosphate (as casein is a phosphorprotein) On heating with NaOH, casein is digested and phosphorus is released as inorganic phosphate. Ammonium molydate reacts with phoshorus in the presence of nitric acid (acidic medium) to form canary yellow precipitate of ammonium phosphomolybdate PRINCIPLE
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  REACTIONS OF ALBUMIN 1.Biuret test 2. Heat and acetic acid test or Heat coagulation test 3. Half Saturation test 4. Full Saturation test 5. Colour Reacions: i. Ninhydrin test ii. Xanthoproteic test iii. Modified Million’s test iv. Hopkin’s and Cole Test (Aldehyde test) v. Sakaguchi test vi. Sulphur test vii. Molish test Egg albumin is a glycoprotein. It contains all the amino acids and also carbohydrates.
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  REACTIONS OF CASEIN Biuret test  Isoelectric Precipitation test  Modified Neuman’s test (Specific for casein)  Colour Reactions: i. Ninhydrin test ii. Xanthoproteic test iii. Modified Million’s test iv. Hopkin’s and cole test v. Sakaguchi test vi. Sulphur test All the color reactions will be positive except the SULPHUR TEST.
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  REACTIONS OF GELATIN Biuret test  Half saturation test  Full saturation test  Color Reactions: i. Ninhydrin test ii. Xanthoproteic test iii. Modified Million’s test iv. Hopkin’s and Cole test ( Aldehyde test) v. Sakaguchi test vi. Sulphur test Expect ALDEHYDE TEST, MILLION’S TEST AND SULPHUR TEST all the other color reactions will be positive because Trytophan is absent in gelatin.
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  REACTIONS OF PEPTONE Biuret test  Half saturation test  Full saturation test  Colour reactions: i. Ninhydrin test ii. Xanthoproteic test iii. Modified million’s test iv. Hopkin’s and Cole’s test v. Sakaguchi test vi. Sulphur test This is formed by partial degradation of proteins. It is a derived protein and relatively low molecular weight compound.