Answered: 3. Isoelectric focusing is a separation…

Biochemistry

9th Edition

ISBN:9781319114671

Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.

Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.

Chapter1: Biochemistry: An Evolving Science

Section: Chapter Questions

Problem 1P

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ANSWER ONLY NUMBER 3

ALL ABOUT BIO CHEM

Transcribed Image Text:1. Determine the isoelectric pH (pI) of the amino acids (a) Met, M, (b) Glu, E and (c) Lys, K. Show your solutions clearly and systematically. Draw the structures (showing the states of protonation) of each amino acid before and after consecutive ionizations. Indicate the pH (i.e., pKa) values at which these ionizations occur. Specify the pKa values you used for the calculation of pl. The pKa values of the amino acids' ionizable groups are listed in the table below. Table 23.2 The pKa Values of Amino Acids pKa a-COOH pKa a-NH3* pKa side chain Amino acid Alanine 2.34 9.69 Arginine 2.17 9.04 12.48 Asparagine 2.02 8.84 Aspartic acid 2.09 9.82 3.86 Cysteine 1.92 10.46 8.35 Glutamic acid 2.19 9.67 4.25 Glutamine 2.17 9.13 Glycine 2.34 9.60 Histidine 1.82 9.17 6.04 Isoleucine 2.36 9.68 Leucine 2.36 9.60 Lysine 2.18 8.95 10.79 Methionine 2.28 9.21 Phenylalanine 2.16 9.18 Proline 1.99 10.60 Serine 2.21 9.15 Threonine 2.63 9.10 Tryptophan 2.38 9.39 Tyrosine 2.20 9.11 10.07 Valine 2.32 9.62 https://wps.prenhall.com/wps/media/objects/724/741576/Instructor_ Resources/Chapter_23/Text Images/FG23_TB02.JPG 2. Paper electrophoresis of amino acids can be carried out on a paper that is saturated with a working buffer. The wetted paper is subjected to an electric field, causing the movement of the amino acids down the length of the paper. The negatively charged amino acids migrate to the anode (+ electrode) while the positively charged amino acids migrate to the cathode (– electrode). Shown below is a set-up for paper electrophoresis (left) and an electrophoretic profile of an amino acid mixture (right). If the amino acids used in this experiment are the same amino acids specified in question number 1, what are the amino acids X, Y, and Z? Explain your answer. The buffer used in this experiment is TRIS buffer pH 8.0. cathode anode X Y Z Anode Cathode

3. Isoelectric focusing is a separation technique that takes advantage of the differences in
isoelectric pH, pl, of polyprotic species like amino acids. A pH gradient is established
in a gel matrix using an ampholyte, with the pH of the gel increasing from the anode to
the cathode. The amino acids move along the gel
depending on their initial ionic state -
happens when they reach the region in the gel where the pH is equal to their pl values.
If the amino acids used in this experiment are the same amino acids specified in question
number 1, what are the amino acids R, S, and T? Explain your answer.
toward the cathode or the anode
until their charges become equal to zero. This
Cathode
2
pH
T
Anode
http://oregonstate.edu/instruct/bb450/450material/OutlineMaterials/07 08ProteinCh
aracterization/15lsoelectricFocusing.jpeg
Isoelectric Focusing

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