Amino acid titration

The titration curve of an amino acid can be analyzed in the same way as described for acetic acid.
1. Carboxyl group dissociation: Consider alanine, for example, which contains an ionizable α-carboxyl and α-amino group. [Note: Its –CH3 R group is nonionizable.] At a low (acidic) pH, both of these groups are protonated (Fig. 1). As the pH of the solution is raised, the −COOH group of form I can dissociate by donating a H+ to the medium. The release of a H+ results in the formation of the carboxylate group, −COO⁻.
This structure is shown as form II, which is the dipolar form of the molecule (Fig. 1). This form, also called a zwitterion (from the German word for “hybrid”), is the isoelectric form of alanine, that is, it has an overall (net) charge of zero.

Figure 1. Ionic forms of alanine in acidic, neutral, and basic solutions.

2. Application of the Henderson-Hasselbalch equation: The dissociation constant of the carboxyl group of an amino acid is called K₁, rather than K_a, because the molecule contains a second titratable group. The Henderson-Hasselbalch equation can be used to analyze the dissociation of the carboxyl group of alanine in the same way as described for acetic acid:

where I is the fully protonated form of alanine and II is the isoelectric form of alanine (Fig. 1). This equation can be rearranged and converted to its logarithmic form to yield:

3. Amino group dissociation: The second titratable group of alanine is the amino (−NH3 +) group shown in Figure 1. Because this is a much weaker acid than the –COOH group, it has a much smaller dissociation constant, K2. [Note: Its pKa is, therefore, larger.] Release of a H+ from the protonated amino group of form II results in the fully deprotonated form of alanine, form III (see Fig. 1).
4. Alanine pK_s: The sequential dissociation of H+ from the carboxyl and amino groups of alanine is summarized in Figure 1. Each titratable group has a pKa that is numerically equal to the pH at which exactly one half of the H+ have been removed from that group. The pKa for the most acidic group (−COOH) is pK₁, whereas the pK_a for the next most acidic group (−NH3 +) is pK₂. [Note: The pKa of the α-carboxyl group of amino acids is ~2, whereas that of the α-amino group is ~9.]
5. Alanine titration curve: By applying the Henderson-Hasselbalch equation to each dissociable acidic group, it is possible to calculate the complete titration curve of a weak acid. Figure 2. shows the change in pH that occurs during the addition of base to the fully protonated form of alanine (I) to produce the completely deprotonated form (III). Note the following:

Figure 2. The titration curve of alanine.

a. Buffer pairs: The –COOH/–COO− pair can serve as a buffer in the pH region around pK1, and the –NH3 +/–NH2 pair can buffer in the region around pK₂.
b. When pH = pK: When the pH is equal to pK₁ (2.3), equal amounts of forms I and II of alanine exist in solution. When the pH is equal to pK₂ (9.1), equal amounts of forms II and III are present in solution.
c. Isoelectric point: At neutral pH, alanine exists predominantly as the dipolar form II in which the amino and carboxyl groups are ionized, but the net charge is zero. The isoelectric point (pI) is the pH at which an amino acid is electrically neutral, that is, in which the sum of the positive charges equals the sum of the negative charges. For an amino acid, such as alanine, that has only two dissociable hydrogens (one from the α-carboxyl and one from the α-amino group), the pI is the average of pK₁ and pK₂ (pI = [2.3 + 9.1]/2 = 5.7) as shown in Figure 2. The pI is, thus, midway between pK1 (2.3) and pK₂ (9.1). pI corresponds to the pH at which the form II (with a net charge of zero) predominates and at which there are also equal amounts of forms I (net charge of +1) and III (net charge of −1).

6. Net charge at neutral pH: At physiologic pH, amino acids have a negatively charged group (−COO−) and a positively charged group (−NH3 +), both attached to the α-carbon. [Note: Glutamate, aspartate, histidine, arginine, and lysine have additional potentially charged groups in their side chains.] Substances such as amino acids that can act either as an acid or a base are defined as amphoteric and are referred to as ampholytes (amphoteric electrolytes).