Isolation of Casein

Protein and Amino Acid Tests


Organic Chemistry 3510

1. Introduction: The purpose of this experiment is to isolate a protein (casein) from a natural source (milk) and to demonstrate that the product obtained is indeed a protein by performing a number of tests on your product.

This experiment is in not in your laboratory textbook. Review the structure and chemistry of proteins and amino acids in your lecture textbook.


Protein Solubility: Proteins are macromolecules which are soluble (suspended?) in water. Proteins are least soluble in water at their isoelectric points and are more soluble at higher or lower pH's. The solubilitiy at pH's different than the isoelectric point appears to be due to the presence of an excess of cationic groups or anionic groups on the surface of the protein. If the protein is, for instance, negatively charged at a pH larger than the isoelectric point, when two proteins bump into each other, the net negative charge repels them and they do not aggregate as one would expect from large molecules. However, at the protein's isoelectric point there is no net charge. When a negative end of one protein bumps into a positive end of another protein, electrostatic attraction causes the two proteins to stick together. Other proteins run into the "dimer" and join the group. Eventually, enough proteins aggregate together that the protein precipitates. In this experiment, casein( pI = 4.6) will be precipitated from milk (pH = ca. 7) by the addition of glacial acetic acid.

Protein Testing:

Biuret Test: The Biuret Test is a general test for proteins. When a protein reacts with copper(II) sulfate (blue), the positive test is the formation of a violet colored complex.

The Biuret Test works for any compound containing two or more of the following groups.

Ninhydrin Test: The Ninhydrin Test is a test for amino acids and proteins with a free -NH2 group. When such an -NH2 group reacts with ninhydrin, a purple-blue complex is formed.

Xanthproteic Test: Phenyl rings containing an activating group can be nitrated producing a yellow product.

The production of a yellow colored product upon the addition of nitric acid is a test for the presence of tyrosine or tryptophan in a protein. The addition of strong base will deepen the color to orange. The yellow stains on the skin caused by nitric acid are the result of the xanthoproteic reaction.

Heavy Metal Ions Test: Heavy metal ions precipitate proteins from their solutions by cross-linking free amino groups and carboxylate groups.

Ions commonly used for testing for the presence of proteins include Zn2+, Fe3+, Cu2+, Sb3+, Ag1+, Cd2+, and Pb2+.

Among the metal ions, Hg2+, Cd2+, and Pb2+ have very high toxicity. They cause serious damage to proteins (especially enzymes) by denaturing them. Victims who have swallowed Hg2+ or Pb2+ ions are often treated with an antidote of a food rich in protein. The protein can combine with the mercury and lead ions and minimize absorption of these ions. Milk and raw egg white are used most often. The precipitated protein complexes are then immediately removed from the stomach by an emetic.

2. Experimental Procedures

A. Isolation of Casein:

a. To a 250-mL Erlenmeyer flask, add 50.0 g of milk (never pour liquids above a balance!!!) and heat (hot plate!) the flask in a water bath (a 600 mL beaker containing about 200 mL of distilled water). Stir the solution constantly with a stirring rod. When the bath temperature has reached about 40o C, remove the flask from the water bath and add about 10 drops of glacial acetic acid while stirring to precipitate the casein.

Filter the mixture into a 100-mL beaker by pouring the mixture through a cheese cloth which is fastened with a rubber band over the mouth of the beaker. (Be sure that the cheese cloth has a small indentation.) After liquid stops coming through the cheese cloth, remove the cheese cloth from the beaker and gently squeeze the cloth to remove most of the remaining water. The filtrate in the beaker may be discarded down the drain. Using a spatula, scrape the precipitate from the cheese cloth back into the Erlenmeyer flask.

Add 25 mL of 95% ethanol to the precipitate in the flask. Stir the mixture for 5 minutes to dissolve most of the fats and water in the mixture. Allow the precipitate to settle and then carefully decant the alcohol solution into another beaker. Discard the alcohol solution in the flammable waste container. Add 25 mL of a 1:1 mixture of ether-ethanol (caution: no flames anywhere!) to the precipitate to remove remaining fat and water. Stir the mixture for 5 minutes and filter the mixture by swirling the flask to create a slurry and then rapidly pouring it into a Buchner funnel under vacuum filtration in a hood. Add about 5 mL of 1:1 ether-ethanol to the flask, swirl the flask and rapidly pour the slurry with the remaining precipitate over the precipitate in the Buchner funnel. Draw air through the precipitate for at least a minute to dry the casein. Weigh the casein and calculate the percent casein in milk.

B. Chemical Analysis of Proteins: For each of the first three sets of tests you will test five different samples. Prepare five clean, labeled test tubes. For each set of tests, place 15 drops of 2% glycine in Tube 1, place 15 drops of 2% gelatin in Tube 2, place 15 drops of 2% albumin in Tube 3, place 15 drops of 2% tyrosine in Tube 4, and place 15 drops of water in Tube 5 along with one quarter of a full spatula of the casein you prepared earlier.

a. Biuret Test: To each of the test tubes, add 5 drops of 10% NaOH solution and two drops of dilute CuSO4 solution while swirling. The development of a purplish violet color is evidence of the presence of proteins.

b. Ninhydrin Test: To each of the test tubes, add 5 drops of of ninhydrin reagent and heat the test tubes in a boiling water bath for about 5 minutes.

c. Xanthoproteic Test: To each of the test tubes, add 10 drops concentrated HNO3 while swirling. Heat the test tubes carefully in a warm water bath. Additionally, add a couple of drops of concentrated nitric acid to a dry piece of hard boiled egg white on a watch glass. Allow the acid to sit on the egg white for at least 5 minutes.

d. Heavy Metal Ion Test: To each of 3 clean, labeled test tubes, add 2 mL. milk. Add a few drops of each of the following metal ions to the corresponding test tubes: Pb2+ as Pb(NO3)2 in test tube 1; Hg2+ as Hg(NO3)2 in test tube 2; and Na1+ as NaNO3 in test tube 3. Dispose of the lead and mercury samples in the appropriate waste containers.

e. Unknown: Determine which one of your group's two unknowns is a nitrogen compound and if it is an amino acid or a protein.

3. Be able to answer the following:

a. How was the casein precipitated?

b. How was the fat removed from the precipitated casein?

c. How was the water removed from the precipitated casein?

d. How does a Buchner funnel differ from a Hirsch funnel? What are the relative advantages of each?

e. Which test(s) differentiate between proteins and amino acids?

f. Which test(s) could be used to differentiate between an amino acid and a monosaccharide?

g. Many stains on one's hands can be removed by washing. Why can't nitric acid stains be removed by washing?

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