On the left in both the chemical reaction and the titration curve, you should imagine that alanine is in a very acidic solution at a pH of about 0. As you move from left to right accross the page, you are adding hydroxide to the solution. This increases the hydroxide concentration and decreases the hydrogen ion concentration. Note that alanine loses protons as you move from left to right. At the first pKa, the alpha-carboxyl dissociates. At the second pKa, the alpha-amino dissociates.

Note that at each pKa, the solution is buffered. That is, it resists changes in pH as hydroxide is added. Also note, that the pI occurs where alanine has no net charge.

formula and graph of titration of alanine with hydroxide.
Chapter 6, Objective 13: "Given any amino acid except cysteine, serine, threonine, and tyrosine, be able to predict the isoelectric point." How do I start? How do I figure this out without a graph to look at?

For this course, there are only four categories of isoelectric points that you need to learn:

  • amino acids without any charged R-group (alanine, glycine, ...)
  • lysine and arginine
  • aspartate and glutamate
  • histidine
These four categories will be covered on four different webpages. Although cysteine, serine, threonine, and tyrosine all dissociate, for the purpose of this course we will ignore them?

The isoelectric point is the pH at which an amino acid or protein has no net charge and will not migrate towards the anode or cathode in an electric field. The charges on any amino acid at a given pH are a function of their pKas for dissociation of a proton from the alpha-carboxyl groups, the alpha-amino groups, and the side chains (R-group). The pKa for the alpha-amino groups and the alpha-carboxyl groups are about 2 and 10 (Figure 6.1). The pKas of the important side chains are shown in Figure 6.9.

You start by having a very rough idea of the structure of the amino acid. What are the acidic groups and what are their pKas. Next, you try to visualize the amino acid fully associated with hydrogen and what the charge on the molecule would be. Next, you visualize removing hydrogen ions by titrating with hydroxide ions. You will remove hydrogen ions from the group with the lowest pKa first and, then from the next higher pKa. Eventually you reach the pI.

Example: Calculate the pI for alanine or any amino acid that does not have a charged R-Group.

At very acidic pHs, the alpha-amino group is in the –NH3+ form and the alpha-carboxyl group is in the COOH form so the alanine has a charge of +1.

As we titrate with hydroxide ions, we remove hydrogen ions. They combine with hydroxide ions and become water. When we reach pH 2, the protons on half the alpha-carboxyl groups are removed. This is not the pI because half of the alpha-carboxyl have a negative charge but all of the –NH3+ have a positive charge. So, the net charge on the alanine molecules is positive.

As we titrate with more hydroxide ions, we reach a point where there are just as many COO- groups as there are –NH3+ groups. This is the pI because the net charge on the alanine molecules in solution is 0. This point is half way between the pKa1 and the pKa2.

If we titrate with more hydroxide ions, we will remove more hydrogen ions from the –NH3+. More and More –NH3+ groups will become neutral –NH2 groups. The alanine molecules in solution will have less positive charges than they have negative charges. Now, they will again migrate in an electric field.

To review, you started with knowing that alanine had two dissociable groups and the pKas for those groups. You know that as you titrate, the molecule will change as follows:
COOH, –NH3+ at a pH below pKa1. The net charge is about +1.
COO-, –NH3+ at a pH between pKa1 and pKa2. The net charge is about 0.
COO-, –NH2 at a pH above pKa2. The net charge is about -1
The only neutral solution of alanine must be when the pH is between pKa1 and pKa2. The pI is half way in between.pKa1 and pKa2