The table at the right gives the molecular weight, diffusion coefficient, and pl
ID: 12516 • Letter: T
Question
The table at the right gives the molecular weight, diffusion coefficient, and pl for three proteins. You are trying to decide the best methods for seperating the proteins from each other. Identify the protein which matches each behaviour or property listed below. Elutes first from a get filtration column Elutes first from a diethylaminoethyle cellulose ion exchange column with a decreasing pH gradient. Elutes first from a carboxymethyl cellulose ion exchange column with an increasing pH gradient. Migrates fastest on SDS gel electrophoresis. Has the largest frictional coefficient. Migrates to the anode upon electrophoresis at pH 4. 9. Migrates to the cathode upon electrophoresis at pH 7. 0. Note: If you answer this question incorrectly, a single red X will apear indicating that one or more of the blanks are incorrect.Explanation / Answer
A). Catalase, because it has the highest MW.
High molecular weight proteins will go down through a column swiftly, while lower molecular weight proteins take more time. This is because the structure of the gel beads within the column excludes molecules that are too big to pass through the bead pores. The sample mix of proteins is added to the top of the column along with a constant flow of buffer solution to keep the proteins moving. As the proteins flow past the beads, the smaller ones are free to enter through the pores. The excluded big molecules flow swiftly around the beads and thus, quickly exit the bottom of the column. The smaller proteins get there eventually, after passing through many pores on many beads.
B). Lysozyme will elute first because below pH 11 it will be mostly positively charged, and thus will be repelled by positively charged diethylaminoethyl cellulose exchange column (DEAE-cellulose).
C). Serum albumin will elute first because above pH 4.8 it will be mostly negatively charged, and thus will be repelled by negatively charged carboxymethyl cellulose gel ion exchange column (CM-cellulose).
For any one protein there will be a pH at which the overall number of negative charges equals the number of positive charges and so it has no net charge. This is its isoelectric point (pI), or more strictly speaking its isoionic point. At this pH the protein will not bind to any ion-exchange resin. Below this pH the protein will have a net positive charge and will bind to a cation exchanger, whereas above this pH it will have a net negative charge and bind to an anion exchanger (our case).
D). Lysozyme because it has the lowest MW and will travel faster then the other two.
E). Catalase, since it has the largest surface area due to large MW (molecular weight).
F).At pH 4.9 Serum albumin will be mostly negative and will move toward anode.
The 'Anode' is the Positively charged Electrode (or Element) that attracts the Negative (-) ions (Anions) in a Circuit or Chemical reaction.
G). At pH 7.0 Lysozyme will be mostly positive and will move toward cathode.
A 'Cathode' is a Negatively charged Electrode (or Element) that attracts the Positive (+) ions (Cations) in a Circuit or Chemical reaction.
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