Laboratory Exercise 5: Tests for Proteins and Amino Acids

Question 1

Proteins, essential constituents of the living cells, are high molecular weight compounds which, upon hydrolysis, produce __.

Answer 1

amino acids

Question 2

In a protein, the amino acids are linked by __.

Answer 2

peptide bonds

Question 3

Common Tests for Proteins

Answer 3

1. BIURET TEST
2. HOPKINS-COLE TEST
3. MILLON’S TEST
4. XANTHOPROTEIC TEST
5. REDUCED SULFUR TEST
6. NITROPRUSSIDE TEST
7. SAKAGUCHI TEST

Question 4

Materials for Exercise 5

Answer 4

• Egg white
• Graduated cylinder (10 ml)
• Graduated cylinder (100 ml)
• Test tubes (35 pcs)
• Dropper (15 pcs)
• Beaker (250 ml, 2 pcs)

Question 5

Test solutions for Exercise 5

Answer 5

A. 10% v/v egg white in water (10 mL egg white + 90 mL water)
B. 1% Tyrosine
C. 1% Tryptophan
D. 1% Phenylalanine
E. 1% Arginine
F. 1% Methionine
G. 1% Cysteine

Question 6

Chemicals for Biuret Test

Answer 6

• 10% (w/v) NaOH
• 0.1% CuSO4 (copper(II) sulfate) solution

Question 7

Chemicals for Hopkins-Cole Test

Answer 7

• Hopkins-Cole Reagent (Glyoxylic acid)
• Conc. Sulfuric acid (H2SO4)

Question 8

Chemicals for Millon’s Test

Answer 8

• Millon’s Reagent

Question 9

Chemicals for Xanthoproteic Test

Answer 9

• Concentrated nitric acid (HNO3)
• 10% (w/v) NaOH (same as in Biuret Test)

Question 10

Chemicals for Reduced Sulfur Test

Answer 10

• 10% (w/v) NaOH (same as in Biuret Test)
• 1% Lead acetate

Question 11

Chemicals for Nitroprusside Test

Answer 11

• 2% sodium nitroprusside
• 10% (w/v) NaOH (same as in Biuret Test)

Question 12

Chemicals for Sakaguchi Test

Answer 12

• 40% (w/v) NaOH
• α-naphthol solution (1% w/v in ethanol)
• 10% sodium hypochlorite

Question 13

a general test for proteins comparable to Molisch’s test for carbohydrates

Answer 13

biuret test

Question 14

In the Biuret test, the compounds having two or more peptide bonds and copper salts in an alkaline solution create a __. In other words, the __ interacts with peptide bonds in an alkaline solution. These ions generate a __ when they react with the __ in the peptide link.

Answer 14

• purple color complex
• cupric ions (Cu2+)
• purple or violet-colored complex
• nitrogen

Question 15

In the Biuret Test, __ is produced in an alkaline medium from copper sulfate from the Biuret reagent (__), the cupric ions of which interact with amide nitrogen, resulting in a violet or purple color complex.

Answer 15

• Cupric hydroxide
• 0.1% CuSO4 + NaOH

Question 16

In the Biuret test, the chelate complex is purple in color due to its capacity to absorb light with a wavelength of __. As a result, the presence of proteins in the analyte is indicated by the development of a purple-colored complex. The amount of peptide bonds that are reacting, and hence the number of protein molecules present in the reaction system, determines the absorbance produced. In the Biuret test, short-chain peptides frequently produce a __ or __ color.

Answer 16

• 540 nm
• blue; pink

Question 17

Hopkins-Cole Test test is a confirmatory test of the presence of __.

Answer 17

amino acid tryptophan (indole group)

Question 18

The Hopkins-cole test is based on the principle that the layering of concentrated __ over a mixture of __, with Hopkins-Cole reagent results in the formation of __ or __ at the interface. The formation of the test color is due to the presence of __.

Answer 18

• sulfuric acid
• tryptophan-containing proteins
• purple or violet color ring
• indole group

Question 19

does not respond to Hopkins-cole test due to absence of amino acid tryptophan.

Answer 19

Gelatin

Question 20

The reagent used in Hopkins-Cole test contains __ (__) which condenses with __ in the presence of __ to form colored complexes (usually a violet interface is observed)

Answer 20

• glyoxylic acid; C2H2O3
• indole derivatives
• strong acids

Question 21

This test is used to detect the presence of phenolic group-containing amino acids like tyrosine.

Answer 21

Millon’s Test

Question 22

In Millon’s Test, the reaction takes place because of the __ and any phenolic compounds that are unsubstituted in the __ such as __, __, and __will give the positive reaction. The phenolic group containing amino acids reacts with __ solution (__) and forms a __ of the mercury-amino acid complex. After that, when __ solution is added and heat it until __ colored complex, __is produced.

Answer 22

• hydroxyphenyl group
• 3,5 positions
• phenol; tyrosine; thymol
• acidified mercuric sulfate
• mercury dissolved in nitric acid
• yellow precipitate
• sodium nitrate
• red
• mercury phenolate

Question 23

The xanthoproteic test is an identification test for the presence of __. Aromatic amino acid (__ and __) reacts with __ and produce __-colored __.

Answer 23

• aromatic amino acids
• tyrosine; tryptophan
• concentrated nitric acid
• yellow
• nitro derivatives

Question 24

In the Xanthoproteic test, the benzene ring of __ and the hydroxyl group of __ are easily nitrated into yellow-colored aromatic compounds (__). The addition of NaOH to this solution results in the __ of the phenolic group which produces an __ color.

Answer 24

• tryptophan
• tyrosine
• xanthoproteic acid
• ionization
• orange

Question 25

Xanthoproteic test
__ possesses an aromatic nucleus but does not give a positive reaction because, in normal conditions, nitration of it is difficult.

Answer 25

Phenylalanine

Question 26

This is a test for sulfur-containing amino acids cysteine and cystine (except for methionine where its sulfur is not reactive), which reacts with lead acetate to form black lead sulfide.

Answer 26

Reduced Sulfur Test

Question 27

Reduced Sulfur Test is a test for __ amino acids __ and __ (except for __ where its sulfur is not reactive), which reacts with __ to form __.

Answer 27

• sulfur-containing
• cysteine; cystine
• methionine
• lead acetate
• black lead sulfide

Question 28

Nitroprusside test is a biochemical test used for the detection of the __ in amino acids or the __ amino acid in a protein. A free __ amino acids reacts with __ in the presence of excess __ and produce a __-colored compound. The __ group plays an important role in this reaction.

Answer 28

• free –SH groups
• cysteine
• sulfhydryl group-containing
• sodium nitroprusside
• ammonium hydroxide
• red
• free thiol

Question 29

a biochemical test used for the detection of the free –SH groups in amino acids or the cysteine amino acid in a protein.

Answer 29

Nitroprusside Test

Question 30

Sakaguchi test is given by __ compounds for example __. Specifically, arginine gives __ color by reacting with __ in presence of oxidizing agent like __ or sodium __.

Answer 30

• guanidine
• arginine
• red
• α-naphthol
• bromine water; hypochlorite

Question 31

In the Sakaguchi test, other than arginine, some __ also give this reaction.

Answer 31

guanidine-containing non-amino acids

Question 32

Procedure: Biuret Test

Thoroughly mix 2 mL of __ and 2 mL of __. Add 1 drop of __ solution (Caution: toxic). Mix thoroughly and note if a __ or __ coloration develops. If not, add additional drops (up to 10 drops) of __, mixing the solution after each addition. A positive test is the appearance of a __ or __ coloration; it may be very pale. Use a white background.

Answer 32

• test solution
• 10% NaOH
• 0.1% CuSO4 (copper(II) sulfate)
• pink; violet
• 0.1% CuSO4 (copper(II) sulfate)
• pink or violet

Question 33

Procedure: Hopkins-Cole Test

Take 1 ml of __ and add 1 ml of __ (Caution: corrosive, irritant) in a test tube and mix it well. In a fume hood, carefully add __ (Caution: corrosive) along the side of the test tube, keeping the tube in an inclined position (do not shake the test tube, while adding the acid in test tube). As a result, __ `appears at the junction of the amino acid solution and the __. The formation of ring confirms the presence of tryptophan amino acid in the test sample.

Answer 33

• sample solution
• Hopkins-Cole reagent
• concentrated H2SO4 (Sulfuric acid)
• purple/violet ring
• concentrated H2SO4

Question 34

Procedure: Millon’s Test

Take 2 ml of __ and add 4 drops of __ in it (Caution: toxic, use the fume hood). Mix well and observe whether a __ forms. Then boil it for 2-5 minutes in a water bath to develop a __ which indicates the presence of protein in the test solution. However, excess of reagent produces __ color which does not indicate positive reaction.

Answer 34

• test sample
• Millon’s reagent
• white precipitate
• red color precipitate
• yellow

Question 35

Procedure: Xanthoproteic Test

After adding 1 ml test sample in dry test tube, add equal amount of __ (Caution: corrosive; use the fume hood) and mix well. __ are formed which are converted into __ color upon heating for few minutes. Allow it to cool for some time and then carefully add 1-2 ml of __. __color is observed if the test is positive.

Answer 35

• conc. HNO3
• White precipitates
• yellow
• 40% NaOH
• Orange

Question 36

Procedure: Reduced Sulfur Test

To 2ml of test sample, add 2 ml of __ and heat carefully in a flame for 15 to 30 seconds (be ready to move the test tube away from the flame when __ occurs). Add 5 drops of __ (Pb(CH3COO)2) solution (Caution: toxic, carcinogenic). If a __ of __ does not form, try boiling the mixture again. The precipitate may be so finely dispersed it looks __.

Answer 36

• 10% NaOH
• foaming
• lead (II) acetate
• black precipitate
• lead sulfide
• brown

Question 37

Procedure: Nitroprusside Test

Initially, take 2 ml test sample in test tube and then add 0.5 mL freshly prepared __ (Caution: toxic) by shaking test tube thoroughly. Add __ dropwise (~0.5 mL). __ color indicates positive test

Answer 37

• sodium nitroprusside
• sodium hydroxide
• Red

Question 38

Procedure: Sakaguchi Test

Prepare mixture of 2 ml test sample and 1 ml of __. After that add 2-3 drops of __ (Caution: toxic, irritant). Mix it well and add 6-8 drops of __ or 2-3 drop of __. Observe __ color at the end, which indicates a positive result.

Answer 38

• 40% NaOH
• α-naphthol
• bromine water
• sodium hypochlorite
• red