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Key%20HW%202%20Summer,%202008

Key%20HW%202%20Summer,%202008 - 1 of 4 Biological Sciences...

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1 of 4 Biological Sciences 102 Answer Key Summer, 2008 K. Hilt Homework #2 1. Amino acid analysis is done on protein “Z”. However, in this case, the student decides to use an anion exchange resin, consisting of polystyrene covalently attached to a quaternary amine (the latter will be positively charged at all pH’s) to separate the amino acids. a) What is the minimum pH that the student should load the unknown amino acids onto this column? Show all calculations. pH > 11.05 b) What type of buffer should they use in the loading step? Describe the various factors that you should pay attention to when selecting a particular buffer for this experiment, and then select an actual compound, using appendix IV in “Biochemical Calculations”. If your buffer choice cannot satisfy all of the parameters that you have chosen, then mention what you would need to change in your experiment. Choose a buffer with a pK a of 11 +/- 1 pH unit. I would suggest that you choose a compound whose acid is “+” and base is “0”, to avoid taking up all of the binding positions on the resin. However, do not choose an amine, if you plan to use ninhydrin to assay the fractions eluting from your column. c) Calculate how you would make up 1500 ml of a 0.10 M solution of your buffer, at the pH you chose in part “a” above, using the pK a you got from appendix IV, in part “b” above. You should be able to do this, using the Henderson-Hasselbalch equation. 2. Let’s say that amino acid analysis of protein “Z” in question 1 above gave the following experimental results: Amino Acid Number of Residues in Protein “Z” Amino Acid Number of Residues in Protein “Z” A 13 B 13 G 12 W 6 P 5 H 12 Z 24 C 4 K 8 V 24 R 12 S 12 L 23 T 14 Y 12 F 13 a) What is the approximate net charge of protein “Z” at pH 7? Use the pK a ’s given in homework #1. Why is this net charge only an “estimate”? Discuss. “–16.4” (The C’s, for example, contribute a “–0.2” charge. Do not forget the N- and C-termini). This is only an estimate because the pK a ’s of the side chain R-groups vary, due to their environment. Note: there is no NH 4 + in the above table. That tells us that this protein had no N or Q in it and that B is D and Z is E. If NH 4 + had been present, we would have to modify our net charge calculation. Both D and E would still be negative at pH 7.
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