Ionization of amino acids is important to understand that helps in clearing the concepts of pka, pI of monoprotic, diprotic and triprotic amino acids.. Amino acids are compounds that contain an amine group, -NH₂, and a carboxylic acid group, -COOH and also side chain “R” group that differs for each amino acid. 

Ionization of Amino Acids

In physiological systems at pH 7.4,  where the pH is almost near neutrality, ionization of amino acids occur, as the amino group of an amino acid will be protonated and the carboxylic acid group will be deprotonated.  This is called the zwitterion form as shown below.

But, in strongly acidic solutions ionization of amino acids occur differently (pH low, therefore high H⁺ concentration) the carboxylic acid group will also be protonated, while in strongly basic solutions (pH high, therefore low H⁺ concentration and high OH^– concentration) both the carboxylic acid group and the amino group will both be unprotonated.

The ionization of amino acids or acid-base behavior of amino acids is best described by the Bronsted-Lowry theory of acids and bases.  A simple amino acid (that does not have an acid or base group in the “R” group) is a diprotic acid in its fully protonated form (NH₃⁺ and COOH); it can donate two protons during its complete titration with a base (NH₂ and COO^–).  The titration with NaOH is a two stage titration represented by the reactions below.

⁺NH₃CH(R)COOH  +  OH^– ®  ⁺NH₃CH(R)COO^– +  H₂O

⁺NH₃CH(R)COO^–  +  OH^–  ®  NH₂CH(R)COO^–  +  H₂O

To understand ionization of amino acids more clearly, initially at a very low pH, the carboxylic group will remain as COOH and amino group will remain as NH₃⁺ because at low pH a large amount of H⁺ in the solution are present e.g., at pH 1. Now if we add base it will going to abstract H⁺ from COOH and NH₃⁺ according to the addition of base. As, at pH of 7 COOH lost its H⁺ but NH₃⁺ group do not lose its H⁺ and if we keep adding the base the point will come that NH₃⁺ group will lose its H⁺  too.

Concept of pKa and pI in amino acids

Titration curves are produced by monitoring the pH of given volume of a sample solution (where ionization of amino acids taking place) after successive addition of acid or alkali. The curves are usually plots of pH against the volume of titrant added or more correctly against the number of equivalents added per mole of the sample

               During ionization of amino acids, at the starting point the acid form predominates (for example CH₃COOH). As strong base is added (e.g. NaOH), the acid is converted to its conjugate base. At the midpoint of the titration, where pH=pK, the concentrations of the acid and the conjugate base are equal. At the end point (equivalence point), the conjugate base predominates, and the total amount of OH added is equivalent to the amount of acid that was present in the starting point. During this process, a buffer system forms and the pH of the system will follow the Henderson Hasselbalch relationship. 

The pK_acid (pKa for the carboxylic acid group) is point where half the acid group has been titrated.  Therefore the equation becomes:

pH = pKa

The titration curve (ionization of amino acids ) of the neutralization of acetic acid by NaOH will look like this:

Determination of pKa values

pKa values can be obtained from the titration data by the following methods:

• The pH at the point of inflection is the pKa value and this may be read directly.
• By definition the pKa value is equal to the pH at which the acid is half titrated.

The pKa can therefore be obtained from the knowledge of the end point of the titration. When the concentration of  the protonated form equals that of the unprotonated form, the ratio of their concentrations equals 1, and log 1=0. Hence, pKa can be defined as the pH at which the concentrations  of the protonated and unprotonated forms of a particular ionizable species are equal. The pKa also equals the pH at which the ionizable group is at its best buffering capacity; that is the pH at which the solution resists changes in pH most effectively.

Therefore it is clear that, when a weak monoprotic  acid (CH₃COOH) is titrated by a base, a buffer system is formed. The pH of this system follows the Henderson-Hasselbalch equation: This curve empirically defines several characteristics (the precise number of each characteristic depends on the nature of the acid being titrated)

• the number of ionizing groups,
• the pKa of the ionizing group(s),
• the buffer region(s).

Based on the number of plateaus on a titration curve, one can determine the number of dissociable protons in a molecule. The one plateau observed when acetic acid is titrated indicates that it is a monoprotic acid (i.e., has only one dissociable H⁺). Many organic acids are polyprotic (have more than one dissociable H⁺).

The protein building blocks, amino acids, are polyprotic and have the general structure. When an amino acid is dissolved in water it exists predominantly in the isoelectric form. Upon titration with acid, it acts as a base, and upon titration with base, it acts as an acid (a compound that can act as either an acid or a base is known as an amphoteric compound).

The majority of the standard amino acids are diprotic molecules since they have two dissociable protons: one on the alpha amino group and other on the alpha carboxy group. There is no dissociable proton in the R group. This type of amino acid is called a “simple amino acid”. A simple amino acid is electrically neutral under physiological conditions. It is also possible to have a simple amino acid which is triprotic.

The order of proton dissociation depends on the acidity of the proton: that which is most acidic (lower pKa) will dissociate first. Consequently, the H⁺ on the α-COOH group, pKa1 (pKa1 values range between 1.8 and 2.8) will dissociate before that on the α-NH₃ group, pKa2 (pKa values range between 8.8 and 10.6). The titration curve for this process looks similar to the following:

This curve reveals the ionization of amino acids , in addition to the same information observed with a monoprotic acid, an additional characteristic of polyprotic acids and that is the pH at which the net charge on the molecule is zero.

Isoelectric Point

As discussed and represented in the curves above that shows the ionization of amino acids, 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. There are only three categories of isoelectric points that we need to understand.

Amino acids without any charged R-group (alanine, glycine etc.)

The first pKa (R-COOH) is 2.35 and the second pKa (R-NH₃⁺) is 9.69. The isoelectric pH (pI) of alanine thus is:

Amino acids with negative charged R-group (aspartate and glutamate)

For polyfunctional acids, pI is also the pH midway between the pKa values on either side of the isoionic species. For example, the pI for aspartic acid is

Amino acids with positive charged R-group (Histidine, Lysine and Arginine)

For lysine, pI is calculated from

When assessing the charge on a structure at physiological pH, the isoelectric point (pI) is a useful reference. The pI is the pH at which the structure carries no net charge.

pKa-and-pI values of different amino acids