On the left in both the chemical reaction and the titration curve, you should imagine that aspartic acid 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 aspartate loses protons as you move from left to right. At the first pKa, the alpha-carboxyl dissociates. At the second pKa, the R-group carboxyl dissociates, At the third 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 aspartate has no net charge.
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
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.
At very acidic pHs, the R-group is in the COOH form, the alpha-amino group is in the –NH3+ form and the alpha-carboxyl group is in the COOH form so aspartate has a net charge of +1
As we titrate with hydroxide ion, 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 alpha-amino groups –NH3+ have a positive charge and all of the R-Group (COOH ) have no charge. So, the net charge on the aspartate molecules is a positive 0.5.
As we titrate with more hydroxide ions, we reach a point half way between pKa1 and pKa2. At this pH, half of the protons have been removed from the two carboxyl groups and half of the carboxyl groups are not dissociated so the net charge on the carboxyl groups is a -1. The alpha-amino group is fully charged so it has a net charge of +1. The net charge on the aspartate molecules is 0. This is the pI
As we titrate with more hydroxide ions, we reach the pKa2, At this pH, all of the protons have been removed from the alpha-carboxyl group and half of the half of the protons have been removed from the R-group carboxyl groups. The net charge on the carboxyl groups is a -1.5. The alpha-amino group is fully charged so it has a net charge of +1. The net charge on the aspartate molecules is -0.5.
As we titrate with more hydroxide ions, we reach the pKa3, a point where all the carboxyl groups are dissociated and only half of the alpha-amino groups still have a positive charge. The net charge is a negative 1.5. We did not have to go this far to determine the pI but I thought it might be useful.To review, you started with knowing that aspartate had three dissociable groups and the pKas for those groups. You know that as you titrate, the molecule will change as follows:
|COOH, COOH, –NH3+||at a pH below pKa1. The net charge is about +1.|
|COO-, COOH, –NH3+||at a pH between pKa1 and pKa2. The net charge is about 0.|
|COO-, COO-, –NH3+||at pH above pKa2. The net charge is about -1.|
|COO-, COO-, –NH2||at a pH above pKa3, the net charge is about-2.|