Transcript Lab Activity 7 Proteins Part I

Lab Activity 7
Proteins
Part I
IUG, 2016
Dr. Tarek Zaida
1
1. Isolation of Casein from
Cow milk
2
Background
Carbohydrates
Water
Lipids
Milk
composition
Minerals
Proteins
Vitamins
3
4
Casein
MW
Phosphate
Kd
groups/molecule
α
27.3
9
β
24.1
4-5
κ
8.0
2
5
Casein can be precipitated by:
1. Calcium ions
2. HCl
3. Renin
4. Bacteria
6
Experiment1: Isolation of Casein from cow milk
•
•
•
•
•
Reagents
20 ml milk,
Glacial acetic acid (100%),
Ethanol (95% v/v),
Ether, Thermometer to 100 oc.
7
Procedure
1. Place 20 ml (20 g) of milk into a 125 ml flask and
heat at 40 oC in a water bath.
2. Add 5 drops of glacial acetic acid and stir for
about 1 min.
3. Filter the resulting mixture through 4 layers of
cheesecloth held in a funnel and gently squeeze
out most of liquid.
4. Remove the solid (casein and fat) from the
cheesecloth, place it into a 100 ml beaker and
add 10 ml of 95% ethanol.
8
5. Stir well to break up the product. Pour off the
liquid and add 10 ml of 1:1 ether-ethanol
mixture to the solid. Stir well and filter
through 4 layers of cheesecloth.
6. Let the solid drain well, then scrape it into a
weighed filter paper and let it dry in the air.
• Calculate the casein percentage in milk as
follows: % Casein =__grams of casein__ x 100
•
grams of milk
9
Color Reactions of Proteins
10
Proteins are….
• The most important cell content after water
• Are either functional or structural
• Macromolecules made up of amino acids,
connected together by peptide bonds.
Peptide bond: Amide bond, formed between
COOH & -NH2 of 2 adjacent amino acids.
11
Amino acids
Proteins are made up of 20 A.A. All of them have the same general
structural formula shown above, however they are different in the
R- group (side chain).
12
Classification of amino acids
• Non-essential..
Are synthesized by the body
• Essential..
(Valine, Leucine, Isoleucine, Methionine, Threonine, Tryptophan,
Phenylalanine, Lysine, Histidine)
13
What amino acids chemical reactions are due to?
1. Amphoteric nature
2. R-group or side chain

The accessibility of certain functional groups to the reagent will
determine the intensity of the product color.
 The color intensity varies among proteins and is proportional to the
number of reacting functional, or free groups present.
14
A.A in acidic, neutral, and basic solutions
15
Experiments
• A.A can be characterized qualitatively by using
several dyes that will react with certain groups
of the A.A.
 Tests:
1. Millon’s
2. Xanthoproteic
3. Hopkin’s- Cole (glyoxylic Acid)
16
1. Millon’s
• Any compound containing a phenolic hydroxyl
group will give a positive result with Millon’s
reagent.
• Cosequently..
• Proteins containing tyrosine will give a
positive test of a pink to dark-red color
• Note: Some proteins will initially form a white
precipitate that will turn red when heated.
17
Procedure..
1. 2 ml of 2% casein, 2% egg albumin, and
0.1 M tyrosine add 3 drops of Millon's
reagent.
2. Immerse the tubes in a boiling water bath
for 5 minutes.
3. Cool the tubes down. Record the colors
formed.
18
2. Xanthoproteic
 For detection of aromatic groups, derivative
of benzene, (hence aromaric amino acids).
These aromatic groups can undergo reactions
characteristic of benzene, and its derivatives.
19
One such a characteristic reaction for benzene
is: Nitration
 The amino acids tyrosine and tryptophan contain activated
benzene rings and readily undergo nitration, while
phenylalanine does not contain a readily activated benzene
ring.
 a. Act. tyrosine
 b. Act. Tryptophan
phenylalanine
20
Procedure...
1. Add 1 ml of a concentrated HNO3 in a test
tube containing 2 ml of a protein solution.
2. The formed white precipitate, will turn
yellow upon heating, and finally will dissolve
giving a yellow color to the solution.
3. Cool the solution down. Carefully add 3 ml
of 6 N NaOH. The yellow color turns orange.
21
3. Hopkins-Cole (Glyoxylic Acid Reaction)
• Specific for tryptophan (the only
amino acid containing indole group)
• Reacting with a glyoxylic acid in the
presence of a strong acid, the indole ring
forms a violet cyclic product.
• The protein solution is hydrolyzed by
conc. H2SO4 at the solution interface.
• Once the tryptophan is free, it reacts with
glyoxylic acid to form violet product.
Indole
Glyoxylic acid
22
Procedure..
1. In a test tube, add to 2 ml of the solution
under examination, an equal volume of
Hopkins- Cole reagent and mix thoroughly.
• Incline the tube and let 5 to 6 ml of conc.
H2S04 acid flow slowly down the side of the
test tube, thus forming a reddish - violet ring
at the interface of the two layers. That
indicates the presence of tryptophan.
23