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E Lab

This document provides information about protein structure and properties, as well as procedures for several common protein tests. It begins with an introduction to protein structure and properties such as denaturation. It then describes the objectives and materials for a series of experiments involving precipitation of proteins using heat, acids, salts, and other reagents. Finally, it outlines procedures for common protein identification tests including the Biuret, Xanthoproteic, Ninhydrin, Millon's, and Hopkins-Cole tests to detect the presence of proteins or specific amino acids.

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Althea Valenzuela
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167 views3 pages

E Lab

This document provides information about protein structure and properties, as well as procedures for several common protein tests. It begins with an introduction to protein structure and properties such as denaturation. It then describes the objectives and materials for a series of experiments involving precipitation of proteins using heat, acids, salts, and other reagents. Finally, it outlines procedures for common protein identification tests including the Biuret, Xanthoproteic, Ninhydrin, Millon's, and Hopkins-Cole tests to detect the presence of proteins or specific amino acids.

Uploaded by

Althea Valenzuela
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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Name Group

ACTIVITY Proteins Section Schedule


5
Instructor Date

ACTIVITY 5
Proteins

INTRODUCTION

Proteins, the biological macromolecules of molecular weight 5000 or greater, are made up of amino acids joined
together by amide or peptide bonds formed between the -amino group of one amino acid and carboxyl group of another.
May proteins also contain non-amino acid group as part of their structures.

Properties of proteins are properties of the peptide and the groups attached to the -carbon atom. The reaction with
Biuret reagent depends on the presence of peptide bonds while the reactions with Xanthoproteic, Millons reagent depends
on the presence of characteristics groups in the protein molecules such as an aromatic ring.
Proteins may also undergo denaturation which structurally is a disorganization of the molecular configuration of a
protein. After denaturation, coagulation usually occurs. Proteins maybe denatured by heat, alkaloidal reagents, heavy metal
salts, and concentrated acid or alkali.

Tests and Reactions


In Xanthoproteic test, the aromatic rings on the side chains of proteins or free amino acids become substituted with
nitro groups when treated with concentrated nitric acid. The nitrated products are yellow both in solution and as precipitate.
When the solution is made with basic sodium hydroxide, the color becomes more intense and the color ranges form yellow
to orange to brown.

The Biuret test is a general test for proteins due to the presence of the peptide bond. The positive result gives a
violet-pink complex due to the coordination of cupric ion with the lone pair of the amide nitrogen and ozygen form water in
basic solution.

The Ninhydrin test involves several complex reaction between the amino group of the alpha carbon and the
ninhydrin solution. Two ninhydrin molecules cause the removal of the alpha amino group from an amino acid or protein to
form a blue-violet complex.

The Hopkin’s Cole test involves the condensation of typtophan to form a violet colored complex upon reaction with
glyoxylic acid and concentrated sulfuric acid due to the presence of the indole ring.

The Millon’s test if positive for proteins containing a phenol group in the side chain such as that of tyrosine. The
positive test results in the formation of a flesh to red colored precipitate which is due to the mercury salt produced in the
reaction.

OBJECTIVES
1. To determine the effects of heat, heavy metals, acid and alcohol on proteins.
2. To observe reactions of proteins on specific tests

MATERIALS
test tube (16), test tube rack, test tube holder, 5-mL pipet, dropper, 600-mL beaker, wire gauze, burner, tripod,
thermometer

REAGENTS
distilled water, egg albumin, gelatine solution, tyrosine, tryptophan, 2% HgCl 2, 0.1M NaCl, 1% AgNO3, tannic acid, 95%
ethanol, concentrated H2SO4, concentrated HNO3, concentrated HCl, ammonium sulphate, 3M NaOH, 3M HCl,
phenolphthalein, methyl orange, 1% CuSO4, ninhydrin solution, Millon ‘s reagent, Hopkins-Cole reagent
1
____________________________________________________________________________________________________Activity 5

PROCEDURE

I. Precipitation of Proteins
A. Action of Heat
1. Place 2 ml egg albumin solution in one test tube to another tube, 2 ml gelatin solution.
2. Place both tubes in a beaker of boiling water for 5 minutes.
3. Record your observation.

B. Action of heavy metal salts


1. Place 1 ml of albumin solution in each of 3 test tubes.
2. Add 1 drop of 2% HgCl2 to the first tube, a drop of 0.1M NaCl to the second, and a drop of 1% AgNO 3 to the third.
Record the results.
3. Repeat the procedure using gelatin solution instead of albumin.

C. Action of Tannic Acid and Picric Acid


1. To a test tube, place 1 ml of albumin solution and to another tube, 1 ml of gelatin solution.
2. Add a few drops of tannic acid solution to both tubes. Record your observation.
3. Repeat the procedure adding picric instead of tannic acid. Observe.

D. Action of Ethanol
1. Label 3 test tubes 1 to 3. To tube 1, place 1ml of 95% ethanol, to tube 2, mix 0.5 ml water with 0.5ml ethanol and
to tube 3, mix 2 ml water with 0.5 ml ethanol. Shake test tube 3 and discard some of the solution so that only 1 ml
remains.
2. To each of the 3 tubes, add 3 drops of egg albumin solution and note any evidence of formation of a precipitate.
Record results

E. Action of Strong Acids


1. Place 1 ml of albumin solution in each of 3 test tubes.
2. To test tube 1, place a few drops of conc. HNO3, to tube 2, add a few drops of conc. HCl, and to test tube3, add a
few drops of conc. H2SO4. Observe.
3. Repeat the procedure using gelatin instead of albumin.

F. Action of Ammonium Sulfate


1. To 3 ml water, add solid ammonium sulfate until a saturated solution is produced.
2. Add 1 ml of egg albumin solution to the saturated solution of ammonium sulfate. Describe the result.

I. Buffering Action (Amphoteric Property) of proteins

1. Place 1 ml distilled water in each of 2 test tubes.


2. To the first tube, add a drop of phenolphthalein and a drop of 3M NaOH. Shake.
3. To the second tube, add 2 drops of methyl orange and a drop of 3M HCl. Shake.
4. Note the colors in each tube.
5. Add 2 ml of albumin solution to both test tubes, stopper and shake. Note any change in color during a 5-minute
period.

II. Reactions of Proteins Used in Analysis and Identification

A. Biuret Test
1. To a test tube, place 3 ml of egg albumin solution.
2. Add 4 ml of 3M NaOH and mix.
3. Add a drop of 1% CuSO4 and shake. If no change in color is noted, add a few more drops of CuSO 4. Note the
result.
4. Repeat the test using glycine solution of egg albumin.

2
_____________________________________________________________________________________________________Activity 5

B. Ninhydrin Test
1. Place the quantities indicated in 4 separate tubes: 2ml albumin, 2 ml gelatin, 3 drops tyrosine, 3 drops tryptophan.
2. To each tube add 0.5 ml ninhydrin solution and heat to boiling for 1 to 2 minutes. Allow to cool.
3. Tabulate your results.

C. Xanthoproteic test
1. Place the quantities indicated in 4 separate tubes: 1 ml albumin , 1 ml gelatin, 3 drops tyrosine, 2 drops trytophan.
2. To each tube, add 5 drops of concentrated HNO3 carefully and mix.
3. Place all tubes in a water bath maintained at 600C water bath.
4. Observe.

D. Millon’s test
1. To the specicified quantities of each of the following substances: 1ml albumin, 1ml gelatin, 2 drops of tyrosine, 2
drops of tryptophan, in separate tubes add 3 drops of Millon’s reagent. Note the colors.
2. Mix the contents of each tube and place all the tubes in a 600C water bath.
3. Observe.

E. Hopkin’s-Cole test
1. Place the specified quantities of each of the following substances in separate tubes: 2 ml albumin, 2 ml gelatin, 2
drops tyrosine, 2 drops tryptophan.
2. To each tube, add 2 ml Hopkin’s-Cole reagent and mix. Then incline the tube and slowly pour down the side 2 ml
of concentrated H2SO4.
3. Note any color that develops at the interface of the two liquids.

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