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LAB 25: PROTEINS
THE CHEMISTRY OF CASEIN AND ALBUMIN
PURPOSE: To isolate Casein (milk protein) the base for glue and paint.
To denature albumin (egg protein).
To perform color tests to identify the presence of proteins.
SAFETY CONCERNS:
Always wear safety goggles. Handle the concentrated acids (hydrochloric, nitric, and sulfuric) with care.
Avoid skin contact. Wash your hands after use.
Ninhydrin may be irritating to skin and mucous membranes.
Most organic compounds, such as ethanol are flammable.
AMINO ACIDS & PROTEINS:
Proteins, essential components of the living cell, are high molecular weight compounds which, upon
hydrolysis, produce amino acids. In a protein the amino acids are linked together by series of peptide
bonds. A peptide bond is an amide linkage; it is the functional group which can be hydrolyzed (split
apart by water).
H H O H H O H H O
H N C C N C C N C C OH
H R
R'
2H2O
H R
H+
H H O
H H O
H N C C OH
R''
+
H N C C OH
H H O
+
H R'
H N C C OH
H R''
Human cannot synthesize ten of the twenty common amino acids found in protein; these essential amino
acids must be supplied in the diet.
The presence of both the basic amino group (NH2-) and the acidic carboxyl group (-COOH) accounts for
the fact that amino acids (or proteins) in water solution can act as either acids or bases. Substances that
can act as either acids or bases are amphoteric. In this experiment you will use indicators
(phenolphthalein and methyl orange) to illustrate the amphoteric properties of casein, the principle protein
found in cow's milk. Methyl orange appears red in a solution having a pH less than 3, and yellow in a
®
solution of pH greater than 4. The indicator phenolphthalein used to be used as a laxative in Ex-Lax ; it
is colorless in a solution of pH less than 7.8 and pink in a solution having a pH greater than 9.6.
ISOELECTRIC POINT:
Amino acids (and proteins) contain basic amino groups; when an amino group accepts a proton in an
acid-base reaction, the compound becomes positively charged. When the carboxylic acid group of an
amino acid (or protein) donates a proton, the substance becomes negatively charged. At a specific pH, the
compound will have a neutral charge (charge = 0); this pH is the isoelectric point of the protein (or
amino acid). Each protein has a characteristic isoelectric point. At this pH, the molecules coagulate and
are removed from solution.
We will analyze Casein, the major storage protein in milk to determine its isoelectric point.
CH106 Lab 25: Proteins (W13)
65
DENATURATION OF PROTEINS:
The primary structure of a protein refers to the exact order in which the amino acids are linked together
by means of peptide bonds. The three-dimensional configuration of segments of the protein chain is
called its secondary structure; common secondary structures are the alpha-helix and the pleated-sheet.
The secondary structure is formed when amino acids hydrogen bond to other amino acids farther along
the polypeptide chain. The tertiary structure is the three-dimensional shape of the entire polypeptide
chain. Globular proteins, for example, are very tightly folded into a compact spherical form. This folding
results from interactions between the R side chains of amino acids, and may involve hydrogen bonding as
well as disulfide bondings, salt bridges, and hydrophobic interactions. Only proteins containing more
than one chain, such as hemoglobin, have a quaternary structure; the manner in which the several
peptide chains fit together describes a protein's quaternary structure.
If the secondary, tertiary, or quaternary structures of a protein are disrupted, the protein loses is biological
activity and is said to be denatured. Denaturation may result in coagulation with the protein being
precipitated from solution. The factors that may cause the denaturation of proteins are pH, heat, certain
organic solvents, heavy metal ions, alkaloidal reagents, reducing agents, and non-ionizing radiation. In
this experiment you will study the denaturation of albumin, the protein found in egg white.
PROCEDURES:
NOTES:
ACTIONS:
1
I. Denaturation of Proteins:
1. Prepare a fresh egg albumin solution by mixing the white from
one egg1 with 200 mL of deionized water.2
This
makes
enough
albumin protein supply for
2 to 3 teams of students.
2
2. Obtain six separate test tubes and label them A through F.
3. Place 2-3 mLs2 of egg albumin solution into each of the tubes.
If needed, filter through
cheesecloth into a beaker
to remove any solid
impurities.
2
4. For each indicated tube perform the following:
A. Heat: Using a test tube holder, heat the egg albumin solution A
over a low flame.
Acid: Add 2 mLs of 10% HNO3 (Nitric acid) to tube B.3
Base: Add 2 mLs of 10% NaOH (sodium hydroxide) to tube C.3
Alcohol: Add 4 mLs of 95% Ethanol (EtOH, C2H5OH) to tube D.3
Heavy Metal Ions: Add 10 drops of 1% AgNO3 (Silver nitrate)
to tube E.3
F. Alkaloids/Tannins: Add 1 mL (20 drops) of 10% tannic acid
solution to tube F.3
B.
C.
D.
E.
5. Describe any changes. Give a brief explanation for the results.
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CH106 Lab 25: Proteins (W13)
The measurement does
not need to be exact. You
could measure one and
eyeball the others to
match.
3
It may be necessary to
stopper the tube and shake
to mix after each addition.
II. Casein Products:
A. Isolation of Casein from Milk:
1. Weigh a 150 mL beaker. Add about 50 mL of nonfat milk to the
beaker and weigh again. Calculate the mass of the nonfat milk
sample.
4
For best result use a pH meter
or indicator paper having
approximately a 4.5 to 8.5 pH
range.
5
It may take from 4-6 mLs.
6
2. Record the pH of the nonfat milk. 4
3. Warm the sample on a steam bath until the temperature of the milk
reaches about 40oC then remove from heat.
4. Add 5% acetic acid (CH3CO2H, vinegar) a little at a time5 with
gentle stirring until the casein (milk protein) becomes insoluble.
When no further precipitation occurs, stop adding acid. 6
5. Determine and record the pH at which the casein becomes insoluble
in solution. This pH is the isoelectric point of casein. 7
6. Separate the solid casein protein from the soluble whey protein by
decanting off the liquid, keeping the solid casein behind in the
beaker.8
7. Wash the solid casein with two 10 mL portions of water. Stirring
and then decanting off the liquid each time to rinse away any
remaining whey protein or acid. Squeeze dry by pressing the protein
between pieces of filter paper or squeezing through cheesecloth.
8. Weigh a watch glass. Transfer the protein to the watch glass and let
the protein dry. Weigh. Calculate the mass of milk protein.
9. Calculate the percentage of casein in the nonfat milk. 9
You’ve made curds and whey
by artificially souring the
milk.
When milk sours
naturally, the souring and
curdling are caused by lactic
acid formed by the action of
bacteria.
7
At the isoelectric point (pI)
the number of positive charges
on a protein equals the number
of negative charges giving it a
net charge of zero. It is at this
pH that the protein becomes
least soluble.
8
Rather than decanting you
could
filter
through
cheesecloth or filter using a
Buchner funnel apparatus.
9
(g’s of solid casein / g’s milk)
x 100 = %
10
If the casein is too clumpy
grind the water and casein
together with a mortar and
pestle to insure a good mix.
11
B. Casein Glue:
10. Mix a chunk of casein10 (about the size of a nickel) with several
drops of water in a small beaker to make a paste.
11. Stir in a pea sized amount of NaHCO3, (sodium bicarbonate, baking
soda) to neutralize any acid remaining from the vinegar and make
the mixture alkaline.11 Watch for bubbles of CO2 gas produced.
Add a little more sodium bicarbonate until no more bubbles appear.
When dissolved in an
alkaline
solution
casein
becomes sticky so can be used
as glue.
12
The glue you made is similar
to non-toxic commercial white
glue. You can make “fancy”
glues of various colors by
adding food coloring.
12. The substance in the beaker is glue.12 Test the adhesive properties
of your product with various materials.
CH106 Lab 25: Proteins (W13)
67
C. Casein Paint:
Pigment Preparation:
NOTES:
1. Prepare paint pigments according to the following instructions using the
13
chart of reagents below to produce the desired colors:
 Place REAGENT A _______ in a small test tube. Add about 3 mLs of
warm water. Shake the tube gently until the reagent dissolves. Can add
a couple more mLs of warm water if needed to dissolve.
 Add REAGENT B _______ to the tube. Stopper the tube and shake it
thoroughly.
 Filter the solution. Discard the liquid filtrate and save the precipitated
pigment on the filter paper.
Pigment
Reactants
Product
White
A. 0.3g CaCl2
B. 0.3g Na2CO3
A. 0.3g K4Fe(CN)6
B. 0.2g CoCl2
A. 0.2g NH4Fe(SO4)2.3H2O
B. 0.2g Na2CO3
A. 0.2g NH4Fe(SO4)2
B. 0.2g K4Fe(CN)6
A. 0.2g NH4Fe(SO4)2
B. 1.0mL sat Na2SiO3
A. 0.2g CoCl2
B. 1.0mL sat Na2SiO3
A. 0.2g CoCl2
B. 0.2g Na2CO3
Add powdered Iron (III) oxide to
casein
Add powdered charcoal to casein
CaCO3
(blackboard chalk)
Co2Fe(CN)6
Green
Brown
Blue (dark)
Orange
Blue (royal)
Lavender
Red
Black
Fe(OH) 3
KFe2(CN) 6
(Prussian Blue)
Fe2(SiO3) 4 + Fe(OH)3
CoSiO3
CoCO3
Fe2O3
C
Paint Preparation:
2. Chop the dried solid casein prepared in Part IA into small pieces to dry.
Then grind it into a fine powder with a mortar and pestle or a kitchen
blender.
3. Place a small amount of ground casein (enough to cover a penny) in an
evaporating dish. Add a few drops of water and mix to make a thick paste.
4. Mix the casein and the prepared color pigment of choice with a wooden
splint or spatula until the desired color is obtained adding more casein or
pigment as needed.
5. Paint a picture.13
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CH106 Lab 25: Proteins (W13)
Casein paints are
water-based
paints
that are inexpensive.
Most
water-based
paints have casein
glue in them to help
them stick to the
walls and ceilings.
LAB 25: PROTEINS:
NAME_____________
DATE______________
PRE LAB EXERCISES:
1.___ At the isoelectric point of a protein,
A. the pH of the solution will be 7
B. the number of positive charges on the protein will equal the number of negative
charges.
C. the pH of the solution will always be greater than 7.
D. the pH of the solution will always be less than 7.
2.___ At the isoelectric point of a protein,
A. the protein will be hydrolyzed into amino acids.
B. the protein will be most soluble in water.
C. the protein will be least soluble in water.
D. the protein will oxidize to form disulfide bonds.
3.___ If 1.5 g’s of casein were isolated from acidification of 50. g’s of nonfat milk what is the
percent yield of casein in the milk sample? (Show your work)
A. 3.0 %
B. 1.5 %
C. 75 %
D. 33 %
E. 0.033 %
4.
After casein is isolated from milk sodium bicarbonate (NaHCO3) is added to neutralize
any acid remaining from the vinegar. Write the balanced equation for the reaction
accounting for the gas produced.
5.
Complete and balance the equation for the double replacement reaction of Cobalt (II)
chloride with Sodium carbonate to form Lavender paint pigment.
CH106 Lab 25: Proteins (W13)
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CH106 Lab 25: Proteins (W13)
LAB 25: PROTEINS:
NAME___________________
PARTNER_________DATE___
REPORT:
I. Denaturation of Proteins:
Protein Used: circle one
Casein Whey
Albumin
Observations
Explanation/Conclusion
(If denaturation occurs explain how/why. Be specific)
A. Heat
B. Acid
C. Base
D. Alcohol
E. Heavy Metal Ion:
Ag1+
F. Tannic Acid
Explanation/Analysis: Why are the results as they are?
Explain any anomalies.
CH106 Lab 25: Proteins (W13)
71
II. Casein
A. Isolation from Milk:
Mass of Milk used
Observations
Initial pH
pI = Isoelectric point
Explanation: Why does adding acid cause a precipitate?
Explain why the most precipitate occurs at the pI.
Mass of casein produced
Percentage of casein in nonfat milk
(show calculation & circle answer)
B. Glue:
Observations
Glue a sample here:
Explanation/Analysis: Why are the results as they are?
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How/why does casein act as glue? Explain any anomalies.
CH106 Lab 25: Proteins (W13)
C. Paint:
Paint pigments made:
Observations
Paint a picture here:
Explanation/Analysis: Why are the results as they are?
Why use casein in paint? Explain any anomalies.
CH106 Lab 25: Proteins (W13)
73
RELATED EXERCISES:
1.___ Precipitation of a protein takes place when
A. the pH is equal to 7 so the protein is neither acidic nor basic.
B. the pH is equal to the isoelectric point (pI) because at this point the protein is negatively
charged.
C. the pH is equal to the isoelectric point (pI) because at this point the protein has no net charge.
D. a solution of protein is hydrolyzed regardless of the pH.
2.___ Paints containing Lead compounds are poisonous because
A. Lead is a heavy metal that denatures life supporting protein enzymes by complexing with SH
side groups and thus changing their shape.
B. Lead catalyzes the hydrolysis of proteins so that life supporting proteins are no longer
available.
C. Lead causes disulfide bridges (S-S) to form in globular proteins and so changes the protein’s
shape.
D. Lead is a heavy metal that causes persons who ingest or breathe them to weigh more.
E. More than one of these.
3.___ Drops of a dilute solution of Silver nitrate (AgNO3) are applied to the eyes of newborn infants
because:
A. Silver is a heavy metal that will denature the baby’s eyes and cause them to open.
B. Silver is a heavy metal that will denature the enzyme proteins of bacteria that may cause
gonorrheal conjunctivitis.
C. Silver is expensive and thus hospitals can charge patients more money.
4.___ Isopropyl alcohol a good disinfectant because
A.
B.
C.
D.
alcohol acts as a solvent to dissolve bacteria.
alcohol denatures the enzyme proteins of bacteria by disrupting the hydrogen bonds.
alcohol hydrolyzes proteins so kills bacteria.
More than one of these.
5.
Tannic acid and picric acid are useful in commercial products for the treatment of burns. Explain
why that may be. (Hint; think about the chemical make up of skin.)
6.
Baby vomit looks and smells similar to Curds and Whey. Explain why.
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CH106 Lab 25: Proteins (W13)