PROTEINS

Human beings require three classes of macronutrients: proteins, carbohydrates, and fats. Of
these three, proteins play a major role in building muscles, cartilage, hair, and skin.

Proteins are the most abundant and functionally diverse molecules in living systems. Virtually
every life process depends on this class of molecules. For example, enzymes and polypeptide hormones
direct and regulate metabolism in the body, whereas contractile proteins in muscle permit movement.
In bone, the protein collagen forms a framework for the deposition of calcium phosphate crystals, acting
like the steel cables in reinforced concrete. In the bloodstream, proteins, such as hemoglobin and
plasma albumin, shuttle molecules essential to life, whereas immunoglobulins fight infectious bacteria
and viruses. In short, proteins display an incredible diversity of functions, yet all share the common
structural feature of being linear polymers of amino acids.

Proteins are high molecular compounds that on acid hydrolysis yield the monomer alpha-amino
acids. An amino acid consists of carboxylic acid that contains the amino group. Each protein molecule is
composed of 20 different kinds of amino acids linked to one another in large numbers and in varying
configurations or structures and sequences. These are linked by a peptide bond. As a consequence,
proteins differ only very slightly from one another; therefore isolating them from one another is quite
difficult from their native form.

About a hundred of different proteins have been isolated in pure crystalline form. All contain
carbon, hydrogen, nitrogen and oxygen; and nearly all contain sulfur. Some proteins contain additional
elements, particularly phosphorus, iron, zinc, and copper. The molecular weights of proteins are very
high, but on acid hydrolysis they all yield a group of simple organic compounds of a very low molecular
weight such as the alpha-amino acids.

There is no simple way of separating and purifying them into a simpler form. A procedure in
separating a certain protein may cause the denaturation or destruction of another of the other protein.
But nonetheless, some simple ways of extracting proteins are guided by some fundamental principle.
 EXPERIMENT No. 8

SIMPLE SEGREGATION OF PROTEINS

OBJECTIVE:

Isolate protein compound on the different samples

MATERIALS:

0.1 M HCl
Skimmed milk
0.01 M Iodine solution
Wheat Flour
1 N Acetic acid
Dried Beans
Mortar and Pestle
Beaker 250 ml, 500 ml
Stirring Rod
Liquid dropper
Graduated cylinder
Filter paper
Litmus paper
Weighing balance

PROCEDURE:

A. Precipitation of Casein from Skimmed Milk

1. Weigh 50 grams of skimmed milk in a 250 ml beaker. Dissolve in a 100 ml of water.

2. Add 0.1 M HCl slowly from the liquid dropper with constant stirring until the pH of the solution
is 5 (use Whatman® pH paper). The acid should be added very slowly not exceeding 5 ml per
minute with constant stirring to avoid extreme acidity.
3. Allow the solution to stand until the precipitated casein settles at the bottom.
4. Decant and discard the supernatant liquid.
5. Wash slowly with water the precipitate until it is no longer acid to litmus paper or to Whatman®
pH paper.
6. Dry the precipitate by pressing between 2 filter papers. Air dry. Weigh the dried precipitate.
7. Note and record your observations on the activity sheet.

B. Separation of Gluten from Wheat Flour

1. To 200 grams of wheat flour, add enough water to make thick dough.
2. Remove excess water and weigh the dough.
3. Knead with your hand in the stream of cold water until all the starch is washed out and
separated. Test the washings with iodine solution drop by drop until no more violet
discoloration on the washings appears.
4. The insoluble material left is the crude gluten, a mixture of several proteins.
5. Weigh a watch glass and place the gluten into it. Air dry and weigh your crude gluten
afterwards.
6. Record all data in the activity sheet and computer for the percentage (%) protein.

C. Separation of Proteins from Beans

1. Measure 2 cups of chosen dried beans and weigh.

2. Soak the beans in the water overnight.
3. After soaking the beans overnight, grind the beans finely using the mortar and pestle while
adding water slowly.
4. Express the milk of the beans by using cheesecloth(katsa).Set aside the residue for checking.
5. Add 1 N acetic acid to the expressed milk until a precipitate appears. Allow to stand for at least
an hour.
6. Strain again thru a cheesecloth and squeeze as much water as you can. A filter paper may also
be used to remove the excess water from the precipitate.
7. Dry your precipitate by pressing it between 2 filter paper .
8. Weigh your dried precipitate. Compute for percentage protein.
9. Note and record the results.
 Name: CHARISMA J. ANTONIO Course/Year/Section: BS PHARMACY 2-1
Instructor: ANGELICA GOMEZ Group No.:________ Date: _________ Score: _______
_______

EXPERIMENT NO. 8

SIMPLE SEGREGATION OF PROTEINS

POST LABORATORY DATA:

CASEIN GLUTEN BEAN PROTEIN

Weight of Sample

Weight of Protein

% Protein by Weight

Draw a flow diagram showing the different steps in extracting proteins from your different samples
and label.

POST LABORATORY GUIDE QUESTIONS:

1. Why do protein form a precipitate when you add an acid to the solution?
 Adding acids to the solution, lowers the pH and leads to positively charging the protein,
due to the proton capture by amino groups. In an aqueous solution the hydration sphere
surrounding a protein is disrupted; occurred imbalance in structure leads to
precipitation.

2. Differentiate the wheat dough from the gluten obtained in terms of weight and physical
characteristics.

 Gluten-free look a little bit different than wheat, it is often denser, tender and even
crumbly than wheat-based and some gluten free flours have a gritty texture. The
browning is also a little bit different.

3. What is purpose of soaking the beans in water?

 The benefits of soaking. Reduces phytic acids and improves the absorption of important
nutrients and minerals, such as protein, iron, zinc and calcium. Reduces tannins and
polyphenols.

4. What predominant proteins are present in beans?

 Globulin and albumin, which make up over 60 and 25% of total mung bean protein,
respectively, represent the main mung bean storage proteins.

5. How were you able to isolate the crude proteins from their main components such as milk,
starch and beans? Discuss briefly.
Commonly used protein separation techniques include the following: ion-
exchange chromatography, affinity chromatography, dialysis, ultrafiltration, size-
exclusion chromatography, electrophoresis [sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE), isoelectric focusing, and capillary electrophoresis.
Casein can be easily separated from milk by making the milk more acidic. This
can be done either by adding acid or by adding bacteria which produce lactic acid.

Proteins obtained from beans can be separated from other components called
protein concentrates and isolates. Bean protein isolation techniques can use extraction
methods with acid-base solvents, salting out methods with salt solvents, and
modification by enzymatic hydrolysis.
LABORATORY DISCUSSIONS:

Isoelectric focussing separates proteins on the basis of their balance of acidic (negatively charged) vs.
basic (positively charged) amino acid residues, which determines a property known as the protein’s
isoelectric point [pI], the pH at which the protein’s net charge becomes exactly zero. Proteins that differ
by as little as one charged residue (e.g., the mono- and di-phosphorylated forms of a given protein) can
be separated using this method. Conversely, however, proteins with very similar pI values but very
different sizes can run at the same position. Two chromatographic methods are frequently used for
protein or (more often) peptide separation. High-performance liquid chromatography (HPLC) can be
used to separate and to purify proteins/peptides based on size, charge or overall hydrophobicity. Thin-
layer chromatography (TLC) can also be used to separate out peptides (e.g., derived from proteolytic
digestion of a protein) based on similar properties. Both of these methods are very useful adjuncts to
gel-based approaches, particularly for peptide analysis and for preparative purposes (e.g., to purify
rather than simply to analyze proteins). Two-dimensional (2-D) gel electrophoresis is a powerful gel-
based method commonly used for ‘global’ analysis of complex samples (i.e., when we areinterested to
characterize the full range of proteins in a sample, not just a few specific proteins). In this technique the
protein is run first in a narrow (often tube-shaped) isoelectric focussing gel, which as already noted
separates proteins on the basis of their isoelectric points (acid vs. basic character). The
isoelectricfocussing gel is then placed over an SDS-PAGE gel and run on the latter in the perpendicular
dimension