Download Proteins Denaturation

Proteins Denaturation
Denaturation of proteins happens when there is a loss in the secondary or
tertiary or quaternary structure of it. Rarely denaturation happens with loss
of primary structure. It happens when there is a loss in stability of the
connecting bonds, for example: when we use denaturated urea. When the PH
is changed at high temperature (usually over 60 C◦) the protein is over
denaturation or at the existence of oxidating or reducing agents, unless the
bonds are present in certain special places. For example: Ribonuclease
enzyme present in the nuclei which contain 124 amino acids and 4
disulphide bonds, when it is exposed to the action of β- mercapto methanol,
some of the bonds are reduced, excess of it will make the protein loose it
folded structure (denaturated), because of the break down of reduced
disulphide bonds.
There are two types of protein denaturation:
1-Reversable denaturation: when the effect of denaturation reagent is
removed by dialysis, the enzyme will gain its activity again. If we store the
protein below 0 C◦, it will loose its activity, once the temperature is
increased the protein is back active.
2-Irreverssable denaturation: if the protein is at temperature over 60 C◦ it
will form an insoluble substances called coagulum as in heating the eggs
white. It cant be returned to its original state.
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Exploring proteins
To explore proteins is to separate and purify them. The techniques used for
exploring proteins are different such as electrophoresis, chromatography and
ultra centrifugation.
Electrophoresis
It is a method or technique used to separate mixture of charged molecules by
moving these molecules through an electrical field through porous
supporting media, the velocity of migration of these molecules depends on
the net charge and on the force of the electric field and friction coefficient.
V= E (net charge) × Z (force of electric field) / f (friction coefficient)
Movement of particles is opposed by two forces, the electrical force and the
friction (viscous drag) which depend on the viscosity of medium and the
mass and the shape of molecules.
The porous supporting media used is of two types:
1-Paper cellulose acetate membrane.
2-Gel type: of many types as: a- Agarose
b- polyacryl amide (PAG).
The function of media is that because it is a porous, therefore it act as a seed
separator so that small molecules moves very fast towards the poles while
the large molecules move at slow rate, and intermediate molecules, velocity
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is intermediate. The molecules with high net charge moves faster and the
molecules with net charge low, their velocity is low.
In gel electrophoresis we use sodium dodecyl sulfate (SDS) to improve the
separation according to the differences of masses.
The sample separate according to size and net charge.
Visulazation is done by staining the membrane (cellulose acetate) by
ponsceaus S stain which gives red color or staining the gel by coomassie
blue or silver dye.
We could differentiate between two different molecules by their relative
mobility. Detection of molecular weight is done by using a standard protein
of known molecular weight, we could calculate the molecular weight of
unknown molecule, the migration depends on size and charge of molecules.
Haemoglobin S: Sickle red blood cell disease.
It is a disease happens due to mutation at a gene causing a change in an
amino acid normally it is glutamic acid replaced by valine. (normally
haemoglobin is HbA).
In Heterozygos state the mutation happens at one chain, therefore it is called
HbSA or HbAS, the patient carries the disease but dose not show it unless
under certain conditions, therefore it is called HbSS, the patient suffers from
haemolysis of RBCs.
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The RBCs have a shape of stickle or cressent like which are easily
haemolysed. This disease is dominant in Iraq in Basrah, Umarah and
Nasriah.
Because there is a mutation of valine instead of glutamic acid, so HbS has
charge different from HbA charge, it is more positive and so it migrate more
faster in electrophoresis. Because it carries higher net charge than HbA.
According to this there will be two different bands, one on the A region, and
one on the S region.
If heterozygous state two bands will appear, if homozygous a single band
only appears. (the test is usually done at PH= 8.6).
The other features of HbS is that the RBCs usually are insoluble or
crystalline under deoxygenated conditions, why?
The substitution of non-polar amino acids (valine) will create such
conformation that make the non polar residues distribute on the out side of
molecules and this causes reduction in solubility of molecules in
deoxygenated state because the presence of non polar molecules will create
sticky ends called sticky patches. When in contact on other deoxy Hb it will
form coagulation and proceeds to form fiber Hb and precipitate inside the
RBCs causing the cressent shape of RBCs.
When RBCs carry HbS enter small capillaries will blocking them, the
process might lead to thrombosis (lessening of blood supply) causing death
after long time.
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Haemoglobin A1c (HbA1c)
It is glycosylated Hb, formed by binding of glucose to α- amino terminal.
Normally there is certain concentration of HbA1c in the blood, increasing
the percentage of HbA1c in blood causes diabetes which means increasing
the level of blood sugar so that the higher blood sugar the higher HbA1c
concentration.
The HbA1c percentage is used to measure the glucose content control in
blood (in PH=6 acidic conditions).
The HbA1c moves faster than HbA in electrophoresis from anode to
cathode.
If we want to purify or separate large amounts of proteins or other
substances, we use the chromatography technique.
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