EXPERIMENT 1: ISOLATION OF INTACT CASEIN
The precipitation of a protein at its
Milk
- Complex biological substance with high
amount of proteins, lipids, and minerals.
- Milk proteins contain all 9 essential amino
acids needed by humans.(Histidine,
isoleucine, leucine, lycine,
methionine,phenylalanine, threonine,
tryptophan and valine.)
- Other proteins in milk: caseins,
lactalbumins, lactoglobulins (casein being the
most dominant)
Proteins
- Naturally occurring, unbranched polymer
made of monomer units called amino acids.
- Chains of amino acids linked by covalent
peptide bonds.
Casein
- Main protein in cows milk
- Classified as a Phosphoprotein
- has phosphate groups attached to the
hydroxyl groups of some of the amino
acid side-chains.
- Exists in milk as a calcium salt (calcium
caseinate)
- Has an isoelectric point of 4.6
- If pH = 4.6, casein has zero net charge, and
its least soluble form as a salt due to
isoelectric precipitation, and can be isolated.
Color Reactions
- Biuret Test :Test for presence of peptide
bonds
- Ninhydrin Test Test for compounds with free
alpha amino group.
- Xanthoproteic Test: test for aromatic amino
acid
- Hopkins-Cole Test: Test for the presence of
indole group in Trp
Why was non-fat milk chosen?
Non-fat milk was chosen due to the fact that
the purpose of this experiment is to isolate
protein.
Choosing milk that contains lipids/fats would
further complicate the process of isolating
protein. Since it would not be removed in the
process of isoelectric precipitation method.
If however, milk with fat was chosen,
centrifugation is a necessary step to separate
the lipids prior to isolating protein.
Isoelectric precipitation method
isoelectric point, at which proteins are
generally least soluble
pI of casein = pH 4.6, pI of milk = approx. pH
6.5
10% HAc is added gradually to lower the milk
pH to 4.6 (pI of casein)
HAc (pKa 4.76) was chosen because of its
common availability and its weakly acidic
properties, so as to not drastically lower the
pH if stronger acids were chosen.
Why does protein precipitate at its isoelectric
point?
At other pH besides its pI, the net charge of
the protein (+ or -) can interact with nearby
water molecules, therefore it is more likely to
dissociate itself with other protein molecules.
Thus, more soluble.
The net negative charge of the micelles that
surround the protein, helps in solubilizing it, in
pH outside pI.
at the pI of the protein, wherein its net charge
is 0, the micelles lose their net negative
charge since their phosphate groups are
protonated.
Once the micelles are neutralized, the protein
is neutralized as well and breaks free from
the soluble solution, it escapes as precipitate.
The average volume used in this experiment
was 1.85 mL of HAc
At its pI, casein starts to solidify, forming a
large amorphous mass
The mass is separated from the remaining
residues (lactose, salts, minerals, and water),
which is discarded, by either filtration or
decantation
The separated mass is then weighed
(average yield of 78%) and subjected to the
color reactions
COLOR REACTIONS
Biuret test
Used to detect presence of peptide bonds (minimum
of 2), positive results yield a violet color. Negative
result yields the initial color. Principle behind is the
reaction of Cupric ions (Biuret reagent) with the
amino groups the amino acid.
- The Biuret test is based on the ability of Cu
(II) ions to form a violet-coloured chelate
complex with peptide bonds (-CONH-
groups) in alkaline conditions.
- More peptide bonds = more concentrated the
color
Ninhydrin test
Used to detect presence of primary amines (all
amino acids except proline)or secondary amines
(proline), positive results yield a violet color.
- The ninhydrin test is positive for amino
acid.
- Free amino acids can react with ninhydrin
reagent yielding a deep purple solution.
- Deep purple solution: RUHEMANNS
PURPLE
- Ruhemanns purple: A blue-violet dye
formed in the reaction of ninhydrin with amino
acids.
- Ninhydrin: oxidising agent.
- causes oxidative decarboxylation of amino
acids
Xanthoproteic test
Used to detect presence of amino acids with
aromatic groups (Phe, Tyr, and Trp), positive results
yield a yellow color. Negative results will yield the
initial color, (usually colorless). Principle behind is
the nitration of the aromatic rings.
- Yellow color: due to xanthoproteic acid
- Xanthoproteic acid is formed through the
nitration of amino acids such as tryptophan.
- Nitric acid is used to be able to nitrate the
amino acids.
Hopkins-Cole test
Used to detect the presence of tryptophan in
proteins, positive results yield a violet interphase
(upon addition of conc. sulphuric acid to form layers)
- Protein solution + Hopkins Cole reagent,
- Hopkins Cole reagent: consists of oxalic
acid.
- Concentrated sulfuric acid: slowly added to
form two layers.
- A purple ring appears between the two
layers if the test is positive for tryptophan
STRUCTURE OF CASEIN
EXPERIMENT #3: PROTEIN ASSAY BY THE
BRADFORD METHOD
Quantification of Protein using Bradford Assay
Bovine Serum Albumin
o is a serum albumin protein derived from
cows. It is often used as a protein
concentration standard in lab
experiments.
o The full-length BSA precursor protein is
607 amino acids (AAs) in length, pH of
1% solution is 5.2-7 .
Coomassie Brilliant Blue G-250
o The different colours are a result of the
different charged states of the acidic dye
reagent.
Principle of Bradford Assay
Proteins containing basic amino acids
(histidine, lysine and arginine) and with
aromatic amino acids (tryptophan, tyrosine
and phenylalanine) bind by hydrophobic or
Van der Waals interaction with Coomasie
Brilliant Blue G-250 dye.
There is an ionic interaction too between the
positively basic amino acid side chains and
the negatively charged sulfonate groups on
the dye molecule.
Protein-Dye Complex causes a spectral shift
from red-brown form of the dye (absorbance
465 nm) to the blue form of the dye
(absorbance at 595 nm)
Linear Regression
To establish if there is a relationship between
concentration and absorbance such that if
one increases the other increases too
Linear Equation: y = mx + b
It is important to know the 2 methods we
learned to determine the concentration of
the unknowns.
The use of the equation for a straight line
which you determined b (using your
calculator) which is the corrected y-axis
intercept (since we are not exactly starting
from zero) and m the slope which is the
gradient. You substituted y (in the
equation above) with the absorbance read
from the spectrophotometer then you
solved for x which is the concentration.
The use of the graph the one you did in
the logbook is important because this
might be the one to be used during the
exams.