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BIOCHEM LAB BSMLS-26 XANTHOPROTEIC TEST What is Xanthoproteic test? •

Xanthoproteic test is for the aromatic amino acids, tyrosine and tryptophan.

Reagents:  

Concentrated nitric acid (HNO3) Sodium hydroxide (NaOH)

Principle •

On addition of concentrated nitric acid, the white precipitate is formed due to denaturation of proteins.

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The phenyl group present in tyrosine and tryptophan undergoes nitration on heating with nitric acid.

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The nitro phenyl group imparts yellow color to the solution.

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The nitro phenyl group imparts yellow color to the solution.

Biuret Test    

Test for proteins A general test for compounds containing a peptide bond (Karki, 2018). A purple-colored reaction is a positive result. The test is positive for compounds with two or more peptide bonds. Since most proteins are tripeptides, they give a positive biuret result.

Procedure – Part 1 1. Mix 1.0 mL of egg white solution and 10 gtts of 6M NaOH in a test tube. -The biuret test requires an alkaline environment to work, thus the addition of NaOH. 2. Add 1 drop of 0.5% CuSO4 solution -Copper (II) sulfate reacts with the protein, and a purple-colored compound is formed. Egg white contains albumin, a protein with more than one peptide linkage, thus giving a positive result for the biuret test. 3. Dissolve one tablet of aspartame or ½ sachet of Equal® in 2.0 mL H2O. -Aspartame (generic) or Equal® (brand name) is an artificial sweetener 4. Add to the resulting solution 10 gtts of 6M NaOH and 0.5% CuSO4 solution. -Aspartame only has one peptide bond, thus giving a negative biuret result (Santiago, n.d.) Biuret reaction (Sundin, n.d.)

Result: The sample was positive for Xanthoproteic test, indicating the presence of aromatic amino acid.

Biuret results (Karki, 2018)

Procedure – Part 2

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Using a clean and dry stirring rod, remove the film and place a small portion of it into a clean test tube.

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Add 5 gtts of ferric chloride solution

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Note the change in the color of the film

1. Add 3 gtts of blood plasma/serum and 3.0 mL of 23% Na2SO4 solution to a test tube. Mix well. -Sodium sulfate acts a precipitant for globulin in the blood (Howe, 1921). 2. Take half of the mixture, and add 1.0 mL ether to it. Centrifuge for 5 minutes. -Petroluem ether acts as a solvent for biological substances (Parasuraman, Siaw Thing, Christapher & So, 2014).

Result 

COLOR OF THE FILM: YELLOWISH WHITE

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POSITIVE RESULT WITH FECL3: RED, PURPLE, BLUE

3. Draw out the lower aqueous layer with a pipet and place it in a test tube. -This layer contains albumin from the serum/plasma. 4. To the remaining half of the first mixture and the albumin, place 1.0 mL of NaOH and 2 gtts of 0.5% CuSO4 -Copper (II) sulfate will react with the globulin to produce a purple compound. This indicates the presence of proteins.

Structure of Casein

Blood serum biuret test result

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It is relatively hydrophobic, making it poorly soluble in water. It is found in milk as a suspension of particles, called casein micelles.

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Caseins are stable to heat treatment.

Ferric chloride test 

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The ferric chloride test is used to determine the presence of phenols in a given sample or compound (for instance natural phenols in a plant extract). Enols, hydroxamic acids, oximes, and sulfinic acids give positive results as well.

Test process 

Place 5 ml of milk on a watch glass and heat over a steam bath until a film is produced on the surface of the liquid.

BETA-LACTOGLOBULIN 

More sensitive to heat treatment than the caseins.

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It can form a layer over the casein micelle at high temperatures.

Conclusion 

The organic derivatives of water, such as alcohols, phenols and ethers have different

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chemical properties. To be acquainted with these properties, certain tests can be made.

liquids. (If no ring is visible, gently agitate the tube to cause a very slight mixing at the interface)

A positive result forms a violet complex with Fe3+, which is intensely colored.

4. Record your observation.

Phenol is acidic, and can form the phenoxide ion. This ion can complex with iron (III) and form a coloured substance. However, in our experiment, the purple color was failed to achieve since evaporated milk was utilized instead of unprocessed milk. With that, it affects the reactivity of the reagent due to the different properties of milk.

Result: Light Purple Ring formation * When gently agitated, a ring formed in the interface of the two liquids as it was mix with the sulfuric acid. Positive Hopkin’s cole test: purple color at the interface. ( tryptophan and egg albumin) Negative Hopkin’s cole test: glycine

Some errors were observed based on the experimental results. This may be due to human errors (choice of milk, ), contaminations, and inappropriate proportions.

Hopkins-Cole Test Principle: 

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The Hopkins-Cole reaction, also known as the glyoxylic acid reaction, is a chemical test used for detecting the presence of tryptophan in proteins. A protein solution is mixed with Hopkins Cole reagent, which consists of glyoxylic acid. Concentrated sulfuric acid is slowly added to form two layers. A purple ring appears between the two layers if the test is positive for tryptophan. Nitrites, chlorates, nitrates and excess chlorides prevent the reaction from occurring.

NINHYDRIN TEST  used as another general test for a-amino acids Ninhydrin (C9H6O4)  a chemical used to detect ammonia or primary and secondary amines. It is a powerful oxidizing agent that reacts to the presence of amino acids which will make the amino acids undergo oxidative deamination

Principle: Procedure: 1. In a test tube, add 2.0 mL of Hopkins-Cole reagent to 2.0 mL of egg white solution and mix thoroughly. 2. Tilt the tube and carefully pour along one side of the tube 1.0 mL of concentrated H SO 3. Hold the test tube in an upright position and observe the color of the ring fed at the interface of the two

 Ninhydrin is a powerful oxidizing agent and in its presence, amino acid undergo oxidative deamination liberating ammonia, CO2, a corresponding aldehyde and reduced form of ninhydrin (hydrindantin).  The NH3 formed from a-amino group reacts with another molecule of ninhydrin and is

reduced product (hydrindatin) to give a blue substance diketohydrin (Ruhemanns complex).  However, in case of amino acid like proline and hydroxyproline, a different product having a bright yellow color is formed.

Sakaguchi Test Principles: •

Sakaguchi test is specific for arginine

•

Sakaguchi test is positive for the amino acid containing guanidinium compound in Arginine.

Reaction: α-amino acids + ninhydrin NH3 + Hydrindantin Hydrindantin + NH3 + ninhydrin product (Rhuemann’s purple)

aldehyde + CO2 +

purple colored Reagents: •

1.0 mL of 10% NaOh solution

•

.2% α – naphthol solution

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sodium hypochlorite (bleach)

 Ninhydrin is reduced to hydindantin during the reaction with the α-amino group.  Hydindantin and ammonia will react with another molecule of ninhydrin to form a purple colored product.

Reaction

Result interpretation: Positive sakaguchi test: Red color ( Arginine) Negative sakaguchi test: no red color ( glycine, albumin) 

Arginine Group that reacts with Sakaguchi test

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The guanidine group in arginine reacts with Sakaguchi reagent to form a red-colored complex.

Conclusion: 

Sakaguchi test is a chemical test used for detecting the presence of arginine in proteins....