Titration curve of amino acids helps us to understand the ionization forms of amino acids at different pH. In this section we will see the titration curve of some amino acids.

Table of Contents

• Titration curve of Alanine
• Titration Curve of Aspartate
• Titration Curve of Lysine
• Titration Curve of Histidine

Titration curve of Alanine

When we start building the titration curve. At very acidic pH, 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 2.4, the protons on half the alpha-carboxyl groups are removed. This is the first pKa of the alpha-carboxyl group. 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⁺. A point will come when NH₃ group is half dissociated known as its pka₂. Further, 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, we started with knowing that alanine had two dissociable groups and the pKas for those groups. We know that as we titrate, the molecule will change as follows:

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.

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.

Titration Curve of Aspartate

Again in this case when we go for building titration curve, also at very acidic pH, the R-group is in the COOH form, the alpha-amino group is in the –NH^₃₊ 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 pKa1and 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, 3.65. At this pH, all of the protons have been removed from the alpha-carboxyl group and half of the half of the protons has 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 of 9.63, 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, we started with knowing that aspartate had three dissociable groups and the pKas for those groups. We know that as we titrate, the molecule will change as follows:

Titration Curve of Lysine

At very acidic pH, the R- group is in the – NH₃⁺form, the alpha-amino group is in the – NH₃⁺ form and the alpha-carboxyl group is in the COOH form so the lysine has a net charge of +2.  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 groups have a negative charge but all of the alpha-amino groups, – NH₃⁺, have a positive charge and all of the R-Group, – NH₃⁺, have a positive charge. So, the net charge on the lysine molecules is a positive 1.5.

As we titrate with more hydroxide ions, we reach the pKa₂ (at pH 10) and the protons on half the alpha-amino groups are removed. This is not the pI because all of the alpha-carboxyl have a negative charge but one-half of the alpha-amino groups have a positive charge and all of the R-Groups (–NH₃⁺ ) have a positive charge. So, the net charge on the lysine molecules is a positive 0.5.

As we titrate with more hydroxide ions, we reach the pI at pH 10.25, half way between pKa₂ (9) and pKa₃(10.5). The protons on half the alpha-amino groups and R-Groups are removed so the sum of their charge is +1. All of the alpha-carboxyl ions have a negative charge so the sum of their charge is -1. So, the net charge on the lysine molecules is 0

As we titrate with more hydroxide ions, we reach the pKa₃, a point where there more COO- groups as there are – NH₃⁺groups. Thus the lysine molecules will have a net negative charge and will migrate towards the positive pole.

To review, we started with knowing that lysine had three dissociable groups and the pKas for those groups. We know that as we titrate, the molecule will change as follows:

The only neutral solution of lysine must be when the pH is between pKa₂ and pKa₃. The pI is half way in between pKa₂ and pKa₃.

Titration Curve of Histidine

Histidine can be used here as an example of the pH-dependence of the net charge of an amino acid. In addition to the carboxyl group and the amino group at the α-C atom with pKa values of 1.8 and 9.2, respectively, histidine also has an imidazole residue in its side chain with a pKa value of 6.0. As the pH increases, the net charge (the sum of the positive and negative charges) therefore changes from +2 to –1. At pH 7.6, the net charge is zero, even though the molecule contains two almost completely ionized groups in these conditions. This pH value is called the isoelectric point. At its isoelectric point, histidine is said to be zwitter ionic, as it has both anionic and cationic properties. Most other amino acids are also zwitter ionic at neutral pH. Peptides and proteins also have isoelectric points, which can vary widely depending on the composition of the

amino acids.

To review, we started with knowing that histidine had three dissociable groups and the pKas for those groups. We know that as we titrate, the molecule will change as follows:

The only neutral solution of histidine must be when the pH is between pKa₂ and pKa₃. The pI is half way in between pKa₂ and pKa₃