OBJECTIVES

 To isolate the following proteins

o Casein from skimmed milk by isoelectric precipitation

o Albumin from skimmed milk by heat denaturation

o Gluten from wheat flour by difference in solubility

o Myoglobin from beef/pork by salt-induced precipitation

 To analyze chemical groups responsible for color reactions and explain the principle involved in each test

 To perform acid, alkaline and enzymatic hydrolysis on the isolated proteins and enumerate the

advantages disadvantages of each type of hydrolysis

 To quantitatively determine the protein concentration in a given sample through Bradford assay

INTRODUCTION
Proteins are naturally occurring, and extremely complex substances that consist of amino acid residues
joined by peptide bond. They are a compound known to be an essential constituent of all cells. Proteins are
present in all living organisms and include many biological compounds such as enzymes, hormones, and
antibodies. Ther are 20 different amino acids commonly found in proteins.

Protein isolation is the process of separating and collecting the purest protein fractions. The procedure
for this process can be viewed as a combination of steps where the protein progresses in purity with each step:
(1) identification and acquisition of a source, (2) extraction from the source, (3) separation from the non-protein
components, (4) concentration of the bulk protein and separation into fractions that contain different proteins,
and (5) fine resolution techniques that provide the final product. Protein isolate is the product that has
undergone isolation. These are the things to be considered in the isolation of proteins from its source:

 Three-dimensional structure of the protein

 Interactions that keep the native conformation of the protein functional

 Acid-base property

 Solubility of proteins in different solvents.

The solubility of the proteins can be altered by changing the pH of the environment. Proteins are
insoluble at their isoelectric pH, the pH at which the net charge is equal to zero. To denature proteins is to
disrupt the native conformation. This can be done by subjecting the proteins to extremes of heat and pH, and
different denaturing solvents. Denaturing alters the protein function, demonstrating the relationship between
structure and function.
 Protein hydrolysis is the process by which peptide bonds are broken under acidic conditions at high
temperatures, generating free amino acid residues. The resulting hydrolysis can then be analyzed by amino acid
analysis to determine the amino acid composition and concentration of proteins.

These experiments aim to isolate the intact protein from different sources by precipitation and by
difference in solubility. The isolated proteins will be characterized qualitatively by calorimetric reactions and
paper chromatography. Spectrophotometric analysis will be used to quantitate proteins in the sample.

Isolation of Proteins
Casein from Skimmed Milk

Procedures:

1. Place 20 g of powdered non-fat milk and 50 mL into a 100-mL beaker. Mix well.
2. Heat the mixture to about 40 degrees Celsius.
*The temperature should not exceed 40 degrees Celsius. Monitor the temperature with a thermometer.
3. When the mixture has reached 40 degrees Celsius, add 10% acetic acid dropwise. Stir the solution gently
after every 5 drops.
4. Continue adding the acetic acid until the pH is 4.6.
5. Filter the congealed casein by vacuum or gravity filtration. Set aside the decantate for the isolation of
albumin.
6. Dry the casein residue and calculate the weight % of the casein isolated from the powdered milk.

Albumin from Skimmed Milk

Procedures:

1. Heat the decantate obtained from procedure 5 of isolation of casein to 75 degrees Celsius for about 5
minutes in a water bath.
2. Decant off the liquid from the precipitated albumin.

Gluten from Wheat Flour

Procedures:

1. Prepare the dough by mixing a cup of wheat flour and enough amount of water.
2. Wrap the dough in cheesecloth and place in a running water until all the starch is washed. Test the
washing with iodide solution until a negative result is obtained.
3. The material is an insoluble crude gluten.

Myoglobin from Muscle

Procedures:

1. Place 6.0 g of minced beef and 6 mL 70% (NH 4)2SO4 solution in a small beaker.
2. Stir the mixture for one minute to release the myoglobin.
3. Express the dark red extract into a new beaker using a cheesecloth.
4. Centrifuge the extract at 13,000 x g for 5 minutes.
 5. Transfer 5 mL of supernatant into another empty centrifuge tube.
6. Add 0.30-0.35 of (NH2)2SO4 crystals ground to fine powder. Mix gently until the solid dissolves. Avoid
frothing.
7. Centrifuge the sample again for at least 5 minutes.
8. Decant off the supernatant. Describe the appearance of the purified myoglobin residue.

Hydrolysis of Intact Proteins

Acid Hydrolysis of Intact Protein

Procedure:

1. Add 5 mL of 6M HCl to 0.5 g isolated protein in a hard glass test tube. Label the test tube.
2. Put a cotton on the test tube to act as a stopper and submit it for autoclaving (15 psi for 5 hrs.).
3. Take note of the appearance of the mixture after autoclaving. Add 10 mL of distilled water and then,
transfer the mixture to a 250 mL beaker.
4. Neutralize the mixture with 1M NaOH. Use the neutralized mixture as a sample for characterization tests
and chromatography.

Alkaline Hydrolysis of Intact Protein

Procedures:

1. Add 10 mL of 4 M NaOH to 0.5 g of isolated protein in a hard glass test tube. Label the test tube.
2. Stopper the tube with cotton and autoclave it for 5 hours in 15 psi.
3. Take note of the appearance of the mixture after autoclaving. Add 10 mL of distilled water and transfer
the mixture into a 250 mL beaker.
4. Neutralize the mixture with 1 M HCl. Use the neutralized mixture as a sample for characterization tests
and chromatography.

Enzymatic Hydrolysis of Intact Protein

Procedures:

1. Prepare 1 g/100 mL distilled water protein mixture.

2. Mix 10 mL of protein mixture and 10 mL of saturated protease solution. Alternatively, 0.5 g of protease
can be added directly to 50 mL protein mixture.
3. Add 10 mL 0.1 M phosphate buffer, pH 7.5.
4. Incubate the tube in a water bath maintained at 35-40 degrees Celsius for 60 minutes.
5. Cool the mixture before using it to the qualitative color reactions tests.
Qualitative Color Reactions

Procedure:

Sample preparation: for each test, prepare in separate test tubes an intact protein solution ( 0.5 g of the protein
in 1mL distilled water) and 0.5 mL of the hydrolyzed sample.

Biuret test

1. Add 2o drops of 2.5 M NaOH to the samples. Mix well.

2. Add 2-3 drops of 0. M CuSO4 solution.
3. Shake the test tube and note the color of the solution.

Ninhydrin test

1. Place 10 drops of 0.1% ninhydrin solution into the diluted samples.

2. Heat the tube in the boiling water bath. Take note of the appearance of a blue violet coloration.

Xanthoproteic test

1. Slowly add 10 drops of conc. HNO3 to the diluted samples. Mix and note the color of the solution.
2. Slowly add 10 drops conc. NaOH. Mix and note again the color of the solution.

Fohl’s test

1. Add 5 drops of 30% NaOH and 2 drops of 5% (CH 3COO)2Pb to the samples.
2. Place the tube in a boiling water bath.
3. Note the appearance of a dark sediment.

Test of Amide

1. Add 1 mL of 20% NaOH to 10 drops of the sample.

2. Place the tube in a boiling water bath.
3. Test for the evolution of gas during heating by placing a moisture litmus paper over the mouth of the
tube. Take note of the result.

Nitroprusside test

1. Add 0.5 mL of 3 M NaOH to 0.5 of the sample.

2. Add 0.25 mL 2% nitroprusside solution.
3. Note the formation of a red solution.
Data and Results

Isolation of Proteins

Proteins Description
Gluten Obtain after washing the dough made with wheat
flour. It is soft and rubbery.
Casein %w/w (25.922 g/20 g) x 100% = 129.61% Insoluble in water,
Albumin Smooth and slippery when touched,
The solution was transparent
Myoglobin Brownish blood-clot like appearance

Hydrolysis of Intact Proteins

Made of Hydrolysis Description of the Hydrolysate

Acidic Slightly cloudy in color
Basic Jelly like mixture was formed at the bottom of the
test tube surrounding the intact protein

Qualitative Color Reactions

Casein and Albumin

Color Reaction Intact Protein Protein Hydrolysate

Acid Base Acidic Basic
Biuret Light blue No change Bluish green Bluish color
Ninhydrin Slightly bluish in No change No reaction Pinkish
color
Xanthoproteic Slightly yellowish No change No reaction No reaction
Fohl’s Turbid whitish color No change Cloudy solution No reaction
Test of Amide Red litmus paper No change No reaction Turned blue
turned blue
Nitroprusside Yellow No change No reaction Golden yellow
Gluten from Wheat Flour

Color Reaction Intact Protein Protein Hydrolysate

Acidic Basic
Biuret Light Purple Clear Violet
Ninhydrin Violet Clear Clear
Xanthoproteic Light Yellow Light Yellow Clear
Fohl’s Cloudy white Clear Clear
Test of Amide Clear before water bath, Light yellow before Light yellow before
Red to Blue (litmus paper) water bath, water bath,
Red to blue (litmus Red to blue (litmus
paper) paper)
Nitroprusside Yellow Clear Rusty brown

Myoglobin from Muscle

Color Reaction Intact Protein Protein Hydrolysate

Acidic Basic
Biuret Green-Brown Clear Purple reddish
Ninhydrin Clear Light yellow Violet
Xanthoproteic Light Yellow Clear Cloudy yellow
Fohl’s Brown Clear Brown
Test of Amide Green before water bath, Clear after water bath Yellow
Black after water bath Red to blue (litmus Red to blue (litmus
Red to blue (litmus paper) paper)
paper)
Nitroprusside Yellow brown Cloudy white Clear