Figure 10.6 Dipolar or zwitterionic form of an amino acid. Amino acids exist in a charged form in aqueous solution, the carboxyl group being dissociated and the amino group associated. Some amino acids also have an extra ionizable group present in their side chain (R group). The ionization of each group is pH-dependent and for each amino acid there is a pH at which the charges are equal and opposite and the molecule bears no net charge. This is called the iso-ionic pH (pi).

> The pKa value expresses the tendency of an acid to dissociate and become charged.

In solution the dissociation of each ionizable group in the molecule may be represented as follows:


The dissociated and undissociated forms of each group exist in equilibrium with each other and the position of the equilibrium (or the tendency of each of the groups to dissociate) may be expressed in terms of the equilibrium (dissociation constant) K, often termed Ka because it refers to the dissociation of groups that liberate protons, i.e. acids. The actual values for Ka are often very small and are conventionally expressed as the negative logarithm of the value, a term known as the pKa value:

This function results in a numerical value which is less cumbersome to use and is comparable with the method of expressing the hydrogen ion concentration of a solution, the pH value:

The concentration of hydrogen ions liberated by the dissociation of an acid is related to the dissociation constant for that acid and this relationship can be expressed by the Henderson-Hasselbalch equation:

where the square brackets indicate the molar concentration of the named substance.

An examination of this equation reveals the fact that when the concentrations of salt and undissociated acid are equal, then the pH of the solution is numerically equal to the pKa for that acid. The lower the value of pKa for an acid, the greater is the ability of the acid to dissociate, yielding hydrogen ions, a characteristic known as the strength of the acid. Amino acids with two ionizable groups, an a-carboxyl and an a-amino group, will be characterized by a pKa value for each group and the actual value will give an indication of the strength of the acidic or basic group concerned.

The ionization of an amino acid is most easily demonstrated in a titration curve, which can be prepared by titrating a solution of the amino acid in the fully protonated form with a solution of sodium hydroxide (Figure 10.7) and plotting the amount of alkali added against the resulting pH of the solution. The titration curve for a simple amino acid will show two regions where the addition of alkali results in only a small change in the pH value of the mixture. The buffering action of an amino acid is most significant over these pH ranges.

The first end-point in such a titration is due to the carboxyl group and the pKa value for this is called pKal while the second pK value is for the amino group and is called pKa2. In practice each acid and its salt will act as a buffer over a pH range of approximately one unit on either side of its pK.d value. For the amino acid alanine, where pKai is 2.4 and pKa2 is 9.6, the most effective buffering action occurs over the pH ranges 2.4 ±1.0 and 9.6 ± 1.0.

In addition to the a-amino and a-carboxyl groups those amino acids with an extra ionizable group will also have a pKd value. Glutamic acid is an example of an amino acid with an extra acidic group (COOH) on the 7-carbon, and lysine is an example of an amino acid with an extra amino group on the e-carbon atom. As a result they each have three ionizable groups and three pKa

form +fi+ form form

ch3 ch3 ch3

Figure 10.7 Titration curve of alanine. A solution of alanine (0.1 mol l"1) in the fully protonated form at pH 2.0 is titrated with 0.1 mol 1~1 sodium hydroxide. The volumes of sodium hydroxide added are recorded and plotted against the resulting pH values to give a titration curve which is typical of an amino acid with only two ionizable groups (one carboxyl and one amino). The two shaded areas show the pH range over which the addition of alkali results in only a very small change in pH and where the amino acid exhibits its most significant buffering action. At a pH equivalent to pifal, there are equal amounts of forms 1 and 2, while at a pH equivalent to pKR2, forms 2 and 3 are in equal concentrations. The pI value for alanine is 6.0 and is the mean of pKa1 (2.4) and pK&2 (9.6). At a pH below its pi value, an amino acid will carry a net positive charge but it will carry a net negative charge at pH values greater than its pi.

Table 10.4

Amino acid

Formula pA"a values

Glutamic acid

(extra COOH group)


(extra NH2 group)

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