MED204, 2019. MOLECULES TO CELLS Wednesday, January 14, 2019 SGD #1: Ionization of amino acids and proteins Prepared by Dr. Pinol-Roma with edits (PC) The goal of this session is to gain a thorough understanding of buffering systems in the body, of the ionization of amino acids, and of how the overall charge of amino acids and peptides is affected under different conditions. In preparation for this session, you should work through the questions below and be prepared to explain and apply your approach to each of them. In addition, you should review the bicarbonate buffering system and its practical implications. We will address some additional scenarios and problems during the session. 1. Use the Henderson-Hasselbalch equation to illustrate what the overall charge of a weak acid would be when the pH equals the pKa, when it is higher than the pKa, and when it is lower than the pKa. 2. A 100 ml solution of 0.1M glycine at pH 1.72 was titrated with 2M NaOH solution. The pH was monitored and the results were plotted on a graph, as shown below. The key points in the titration are designated A through E. For each of the statements (a) to (o), identify the appropriate key point in the titration and justify your choice: (a) Glycine is present predominantly as the species +H3N—CH2—COOH. (b) The average net charge of glycine is +1/2 (c) Half of the amino groups are ionized. (d) The pH is equal to the pKa of the carboxyl group. (e) The pH is equal to the pKa of the protonated amino group. (f) Glycine has its maximum buffering capacity. (g) The average net charge of glycine is zero. (h) The carboxyl group has been completely titrated. (i) Glycine is completely titrated (second equivalence point). (j) The predominant species is +H3N—CH2—COO(k) The average net charge of glycine is –1. (l) Glycine is present predominantly as a 50:50 mixture of +H3N—CH2—COOH and + H3N—CH2—COO(m) This is the isoelectric point. (n) This is the end of the titration. (o) These are the worst pH regions for buffering power. 3. What is the isoelectric point of Histidine? Of Glutamate? You should be prepared to explain how you would determine this value theoretically for each amino acid, based on the pKa values of the relevant groups. The relevant pKa values for Histidine are: pK1 = 1.82; pK2 = 9.17; pKR = 6 For Glutamate, they are: pK1 = 2.19; pK2 = 9.67; pKR = 4.25 4. A peptide has the sequence: Glu-His-Trp-Ser-Gly-Leu-Arg-Pro-Gly (a) what is its overall net charge at pH 3.1 and 11? [you do not need to be precise for this; just use best integer approximation. 5. What is the approximate molecular mass of a protein with 750 amino acids?