Transcript Precipitation of Proteins at isoelectric Point

Precipitation of
Proteins at isoelectric
Point
• Objectives:
• General information about proteins .
• To understand the Isoelectric Point .
• To find the PI of Casein protein .
Proteins
• 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.
Proteins
Protein functions
• 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.
Protein structure
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Primary structure
Secondary structure
Tertiary structure
Quaternary structure
Introduction
• Properties of amino acids in proteins and
peptides are determined by the R 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.
Precipitation of Proteins at isoelectric
Point
Protein solubility
• 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 (Valine, Tyrosine, Tryptophan, Proline,
Phenylalanine, Methionine, Leucine, Isoleucine, Cysteine and Alanine )
• Charged and polar surface residues interact with
ionic groups in the solvent and increase solubility.
• 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.
Principle
• 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.
PH = Pka + log{ (casein acetate sodium )÷
(acetic acid)}
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
Notes
• 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.
Experiment
precipitation of proteins
Procedure
1. Into a 50 ml volumetric flask add 20 ml of
water.
2. Add 0.25 g of pure casein, followed by the
addition of 5 ml of 1 N NaOH solution.
3. 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. Setup a series of 9 test tubes to identify the best
tube that have the most precipitate PI .
5. In the first test tube put 3.2 ml 1 N CH3COOH,
and 6.8 ml H2O and mix thoroughly.
6. In each of the other test tubes (2-9) put 5 ml
H 2O d .
7. From the test tube 1 transfer 5 ml to the test tube
2, and mix thoroughly.
8. 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).
Results
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Results Sheet
Results:
PKa = 4.5
Use the following to indicate the precipitate:
- no precipitate
+ few ppt
+ + Moderate ppt
+ + + maximum ppt
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Comment your results :
in tubes 1.2:
in tubes 3.4.5.6 :
in tube 7: