Transcript Protein structure

 General
information about proteins.
 To understand the Isoelectric Point.
 To find the PI of Casein protein.
Proteins are polymers consisting of 20 kinds of
amino acids.
 Are substance of high molecular weight from
5000 to1000,000 daltons.
 All protein Contain C, H, O, N, and most contain
sulfur, some contain phosphorus and a few have
mineral elements such as Fe, Mg and Cu.
 Serve as structural components of animals

Structure, some proteins provide structural
support collagen, hair, crystallins(eyes).
 Transport, some proteins are responsible for
the transportation of smaller molecules from
one part of the body to another, transport
across cell membranes, etc . An example is
hemoglobin, which transports oxygen from the
lungs to cells throughout the body.

Catalysis enzymes, catalyze the chemical
reactions that allow cells to function.
 Storage, Myoglobin is an example of a storage
protein. Myoglobin stores oxygen in muscles so
that during exercise a ready supply of oxygen is
available in the muscle tissue.
 Hormones, some hormones are proteins, insulin
is an example. Hormones serve as chemical
messengers, carrying signals from one part of
the body to another.

Primary structure
 Secondary structure
 Tertiary structure
 Quaternary structure

Properties of amino acids in proteins and
peptides are determined by the R group but
also by the charges of the titratable group.
 Important to know which groups on peptides
and proteins will be protonated at a certain pH.
 Protein molecules carry charges according to
their amino acid sequence and the aqueous
solvent PH they’re dissolved in.

There are many factors that contribute to protein
solubility.
 The most important determinant its electrostatic
charge.
 The solubility of proteins in aqueous buffers
depends on the distribution of hydrophilic and
hydrophobic amino acid residues on the protein’s
surface. Proteins that have high hydrophobic
amino acid content on the surface have low
solubility in an aqueous solvent.

Hydrophilic amino acid like: (Arginine, Asparagine,
Aspartate, Glutamine, Glutamate, Histidine, Lysine,
Serine and Threonine)
 hydrophobic amino acid are (Valine, Tyrosine,
Tryptophan, Proline, Phenylalanine, Methionine,
Leucine, Isoleucine, Cysteine and Alanine )
 The net charge of a protein molecule is the
arithmetic average of all charges. At a certain
solvent PH the protein net charge will be zero this
is called the isoelectirc point.

At a solution PH that is above the PI the surface of
protein is predominantly negatively charged and
therefore like charged molecules will exhibit
repulsive forces.
 Likewise the surface of the protein is predominantly
positively charged at a solution PH that is below the
PI, and repulsion between proteins occurs, so
protein will be soluble at this PH.
 However, at the PI the negative and positive charges
are eliminated, repulsive electrostatic forces are
reduced and the dispersive forces will cause
aggregation and precipitation.
 The PI of most proteins ranges between the PH 4
to 6.


The phenomenon of precipitation or coagulation
of milk protein casein at low PH as milk
becomes spoiled is one of the common examples
of protein isolation due to changes in the PH.


Using acetate buffer of different PH values to find the
isoelectric point of casein
Can be obtained by determining the PH where
minimum solubility.
› The PH of any solution can be calculated from
Handersonhasselbalch equation:
Maximum
precipitation can be obtained at the isoelectric
point by addition of some reagents such as, ethanol which
dehydrates the molecule and allow neutralization of charge
Proteins tend to aggregate and precipitate at their
pI because there is no electrostatic repulsion
keeping them apart.
 Proteins have different pI because of their different
amino acid sequences (i.e.,relative numbers of
anionic and cationic groups), and thus they can be
separated by adjusting the pH of a solution. When
the pH is adjusted to the pI of a particular protein it
precipitates leaving the other proteins in solution.

volumetric flask
Protein(casein)
1N Acetic Acid
9 Test tubes
1N NaOH
H2O
1.
2.
3.
Into a 50 ml volumetric flask add 20 ml of
water.
Add 0.25 g of pure casein, followed by the
addition of 5 ml of 1 N NaOH solution.
Once casein is dissolved, add 5 ml of 1 N acetic
acid solution, then dilute with H2O to 50 ml
and mix well. The resulted solution is a 0.1 N
casein acetate sodium.
4.
5.
6.
7.
8.
Setup a series of 9 test tubes to identify the
best tube that have the most precipitate PI .
In the first test tube put 3.2 ml 1 N
CH3COOH, and 6.8 ml H2O and mix
thoroughly.
In each of the other test tubes (2-9) put 5 ml
H 2O d .
From the test tube 1 transfer 5 ml to the test
tube 2, and mix thoroughly.
Repeat step 7 for the rest of test tubes (3 9).
9.
Now to each test tube (1 -9) add 1 ml of the casein
acetate sodium solution, and shake the test tubes
immediately.
10.
Let the samples stand for 30 min, and note the
turbidity in the 9 test tubes.
11.
Use )+( and )– (signs to describe the turbidity in
the different test tubes.
12.
You should observe the most precipitation in the
test tube which has the pH around 4.7 (close to the
isoelectric point of casein).
Volumetric flasks are used to prepare various kinds
of solutions the neck is narrow so that slight errors
in reading the meniscus results in relatively small
volumetric differences minimizes volumetric
differences or errors.
 A volumetric flask is used to make up a solution of
fixed volume very accurately.

Results Sheet
 Results:
 PKa = 4.5
 Use the following to indicate the precipitate:
 - no precipitate
 + few ppt
 + + Moderate ppt
 + + + maximum ppt
