* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Proteins Denaturation
Expression vector wikipedia , lookup
Magnesium transporter wikipedia , lookup
Genetic code wikipedia , lookup
G protein–coupled receptor wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Agarose gel electrophoresis wikipedia , lookup
Metalloprotein wikipedia , lookup
Multi-state modeling of biomolecules wikipedia , lookup
Interactome wikipedia , lookup
Biosynthesis wikipedia , lookup
Amino acid synthesis wikipedia , lookup
Point mutation wikipedia , lookup
Two-hybrid screening wikipedia , lookup
Photosynthetic reaction centre wikipedia , lookup
Signal transduction wikipedia , lookup
Protein structure prediction wikipedia , lookup
Nuclear magnetic resonance spectroscopy of proteins wikipedia , lookup
Protein purification wikipedia , lookup
Protein–protein interaction wikipedia , lookup
Gel electrophoresis wikipedia , lookup
Proteolysis wikipedia , lookup
Western blot wikipedia , lookup
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. 1 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 2 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. 3 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. 4 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. 5