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In the case of lysine, which is a basic amino acid, the ionisation pattern is different

again and its isoionic point is the mean of pKa 2 and pKa 3 :

COOH COO–

CH CH CH

Cation

(2 net

positive

charges)

Cation

(1 net

positive

charge)

COO–

Zwitterion

pH 3.0

(isoionic

point)

(CH 2 ) 4 (CH 2 ) 4 (CH 2 ) 4

NH 3

+

NH 3 NH 2 NH 2

+

NH 3

+

NH 3

+

NH 3

+

CH

COO–

Anion

(1 net

negative

charge)

(CH 2 ) 4

NH 2

+

pKa 1 pKa 2 pKa 3

2.2 9.0 10.5

As an alternative to possessing a second amino or carboxyl group, an amino acid

side chain may contain in the R of the general formula a quite different chemical

group that is also capable of ionising at a characteristic pH. Such groups include a

phenolic group (tyrosine), guanidino group (arginine), imidazolyl group (histidine)

and sulphydryl group (cysteine) (Table 8.2). It is clear that the state of ionisation of the

main groups of amino acids (acidic, basic, neutral) will be grossly different at a

particular pH. Moreover, even within a given group there will be minor differences

due to the precise nature of the R group. These differences are exploited in the

electrophoretic and ion-exchange chromatographic separation of mixtures of amino

acids such as those present in a protein hydrolysate (Section 8.4.2).

Proteins are formed by the condensation of thea-amino group of one amino acid

with thea-carboxyl of the adjacent amino acid (Section 8.2). With the exception of

the two terminal amino acids, therefore, thea-amino and carboxyl groups are all

involved in peptide bonds and are no longer ionisable in the protein. Amino, carboxyl,

imidazolyl, guanidino, phenolic and sulphydryl groups in the side chains are, how-

ever, free to ionise and of course there will be many of these. Proteins fold in such a

manner that the majority of these ionisable groups are on the outside of the molecule,

where they can interact with the surrounding aqueous medium. Some of these groups

are located within the structure and may be involved in electrostatic attractions that

help to stabilise the three-dimensional structure of the protein molecule. The relative

numbers of positive and negative groups in a protein molecule influence aspects of its

physical behaviour, such as solubility and electrophoretic mobility.

The isoionic point of a protein and its isoelectric point, unlike that of an amino acid,

are generally not identical. This is because, by definition, the isoionic point is the pH at

which the protein molecule possesses an equal number of positive and negative groups

formed by the association of basic groups with protons and dissociation of acidic

groups, respectively. In contrast, the isoelectric point is the pH at which the protein is

electrophoretically immobile. In order to determine electrophoretic mobility experi-

mentally, the protein must be dissolved in a buffered medium containing anions and

cations, of low relative molecular mass, that are capable of binding to the multi-ionised

protein. Hence the observed balance of charges at the isoelectric point could be due in

303 8.1 Ionic properties of amino acids and proteins