Darkthorn’s Blog

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1. Describe cationic/anionic proteins and the concept of pI.

· Cationic proteins are positively charged because of their protonated side chain groups and NH3 N-terminus (present as NH4+). Anionic proteins are negatively charged because of their de-protonated side group and COOH C-terminus (present as COO-). At different pH’s, proteins can be positively or negatively charged. The isoelectric point is the pH at which a particular protein has no net charge because the overall number of negative charges equals the number of positive charges.

1. Describe the principles of:
   1. Ammonium sulphate precipitation. What was the range of Ammonium sulphate required for your protein? Was there a good yield of your protein?

· 50% concentration of ammonium sulphate was required to produce a good yield of protein (27.2%)

· Ammonium sulphate precipitation relies on the property of proteins to have variable ionic strength (according to the salt concentration) and therefore variable solubility. At low concentrations of salt, the solubility of the protein increases with the salt concentration. However, eventually a turning rate is reached, and as the salt concentration continues to rise the solubility of the protein begins to decrease. Ammonium sulphate precipitation exploits this to almost completely precipitate the protein out of solution when a sufficiently high ionic strength has been reached.

· Process: this process is carried out between 0 – 4*C to maximise protein stability. The solution of proteins is stirred continuously with small aliquots of crushed, solid ammonium sulphate being added in increments. Time is allowed between each increment to allow the ammonium sulphate to completely disperse and react with the required protein. The protein is then incubated at 0 – 4*C to allow the protein to precipitate and then subjected to a centrifuge to separate the precipitate. Each precipitate is then dissolved in fresh buffer to determine the total protein content and the enzyme activity. This is repeated until the percentage enzyme activity in precipitate is greater than 90%.

1. 1. Dialysis

· Is used to remove residual ammonium sulphate from the enzyme solution. The concentrated protein solution is placed in a dialysis bag that is semi-permeable to water and salt, but will not allow the protein to pass out. The bag is then placed in a large volume of buffer so that the salt ions diffuse out to equilibrate the solution. This process is repeated several times until most of the salt has diffused out of the bag and only the protein and the buffer solution are left.

1. 1. Ion-exchange chromatography. What is the principle of separation on the DEAE cellulose? Why was pH 8.0 chosen?

· Uses an insoluble matrix with charge groups covalently attached. Negatively charged exchangers bind positively charged ions (cations). They bind one kind of cation, but then when another, more highly charged cation is introduced, this new cation exchanges with the first.

· Proteins are charged molecules because several of the side chains are ionisable (such as the R-groups of lysine or glutamic acid, and the N-terminal amino acid and C-terminal carboxyl groups present in all amino acids). The charge on the protein depends on the number and type of ionisable amino acid side chain groups. Different proteins have different amino acid compositions and so will have different charges at different pH – allowing them to be fractionated. At the isoelectric point of the protein, it will not be attracted to the ion-exchange resin as there will be no net charge. The ion-exchanger used depends on the pH range of the protein. If the protein is most stable at pH values above its pI, an anion exchanger would be chosen. The pH chosen is crucial because it will determine the charge on the proteins being separated.

· DEAE cellulose contains an ionisable tertiary amine group, giving it a positive charge at pH 7, an anion exchanger. As the pH is lowered, COO- groups become protonated and lose their charge, decreasing their affinity for the resin. By slowly reducing the pH using a buffer pH gradient, the proteins are eluted as relatively clear fractions that can then be compared to the enzyme activity to further extract the protein.

· Process: the ion-exchange resin is poured into the column and then washed with buffer solution before the protein mixture is applied. Proteins that are oppositely charged to the resin will flow straight through the column and not bind. The proteins will be bound to different points along the column, depending on their differences in net charge.

· pH 8 is used because the value should be one pH unit above or below the pI of the protein to be separated to ensure adequate binding (and also so that there is movement from the origin). Also, as DEAE cellulose is an anionic exchanger, the starting pH must be sufficiently high to give a good spread of decreasing pH values (without becoming too acidic).

1. 1. Gel filtration

· Biological molecules can be separated due to the differences in shape and size, which lead them to have different abilities to penetrate porous matrixes. In protein purification, the matrix generally consists of porous beads of an inert, highly hydrated gel.

· This exercise used Sephadex G100 which can separate proteins of approximately 4000 – 150000 molecular weight. Ideally, the gel pore size should allow the desired protein to be partially excluded from the gel beads.

· Process: The gel beads are poured into a glass or plastic chromatography column, then they are allowed to settle by gravity. The column is then washed and brought to equilibrium with buffer solution before the protein (present in more buffer solution) is applied to the top of the column. As the proteins pass down the column they penetrate the pores of the gel matrix to different extents and so travel at different speeds. The larger proteins (that exceed the maximum size of the pores) will pass out first as they are unable to enter the beads. Other proteins will spend a proportion of their time within the gel beads, thereby moving more slowly through the column. The eluate is collected in a series of fractions according to size. The column can be calibrated with standard proteins of known molecular weight, thus giving approximate molecular weights (via graph) to the proteins present in the solution.

1. 1. Electrophoresis

· Relies on the fact that proteins have a net charge at any pH besides their pI. Proteins can be separated because every protein has a different net charge, and so will migrate at different rates towards the electrodes. The extent of molecular sieving relies on the gel pore size of the polyacrylamide gel. Bigger molecules move more slowly through the gel.

· The mixture of molecules to be separated is then placed in wells on a flat slab gel (0.75 – 1.5 mm thick) at a suitable distance from the electrodes so that proteins of different sizes separate out. After this, the positions of the separated protein bands are revealed by staining (Coomassie Blue or a silver stain).

· The main advantage of using electrophoresis is that many protein samples can be separated at once, using identical conditions so that the band patterns produced can be easily and relatively accurately compared.

1. 1. SDS-PAGE

· Uses almost identical principles as electrophoresis with a few minor differences.

· The protein mixture is first denatured by heating at 100*C for 2 – 5 minutes in the presence of SDS and thiol reagent. The mixture of molecules to be separated is then placed in wells on a flat slab gel (0.75 – 1.5 mm thick), which contains SDS and more electrode buffer, at a suitable distance from the electrodes so that proteins of different sizes separate out. After this, the positions of the separated protein bands are revealed by staining (Coomassie Blue or a silver stain).

· The charges of the polypeptides are basically negated by the negative charges provided by the bound detergent, so the SDS-polypeptide complexes have essentially the same overall charge. This means that the polypeptides separate strictly according to size, and hence the molecular weight can be estimated by creating a standard curve of polypeptide molecular weight verses distance migrated of each standard molecular weight marker and comparing the distance migrated by the unknown polypeptide.

1. 1. IEF (isoelectric focusing)

· Separates molecules by their charge characteristics. The protein mixture is subjected to an electric field in an inert support (usually either a vertical polyacrylamide rod gel or horizontal polyacrylamide rod gel) in which a stable pH gradient has been generated. The anode region is at a lower pH than the cathode, and it is ensured that the protein for separation has its isoelectric point within this range. A protein in a pH region less than its pI (therefore postively charged) will migrate towards the cathode. Proteins are separated by their pIs on the gel. It is important to have no molecular sieving effects (by using a large pore gel), so that separation is purely on the basis of charge.

· The stable pH gradient is formed by including a mixture of synthetic, aliphatic polyaminopolycarboxylic acids in the inert support. These are available commercially with either a wide pH range (3 – 10) or a narrow pH range (7-8).

· Unlike SDS-PAGE and electrophoresis, IEF does not provide information about the molecular weights of the separated polypeptides, but is able to provide an estimate of the pI of the polypeptide.

1. 1. 2D-PAGE

· Separates proteins on the basis of charge in one dimension and then by size in another dimension. The separation by charge is carried out by electrophoresis in a rod polyacrylamide gel. The rod is then placed on the top edge of the slab gel, where the proteins then migrate into the gel slab according to size. The gel is then stained so that the proteins appear as dots.

· Is used as a test of purity, but is not always accurate. Sometimes polypeptides will migrate as an aggregate because of interactions with each other. In order to separate out these proteins, additional reagents (such as urea) may need to be used during electrophoresis and sample preparation.

1. Do all the methods give identical values for the molecular mass of a particular protein? Compare the degree of accuracy that can be obtained with the molecular mass determined for SDS-PAGE with that obtained with mass spectrometry.

· The above methods do not give identical values for the molecular mass of a protein. In fact, techniques such as isoelectric focusing do not provide a means for estimating molecular mass (only pI). The molecular mass determined by mass spectrometry is relatively more accurate than SDS-page. SDS-PAGE only estimates the molecular mass by comparing the unknown protein’s distance travelled with a standard log curve created by plotting the values of know molecular mass samples. Mass spectrometry uses the mass to charge ratio of ions to determine the molecular mass of the unknown sample.