9780521516358book.pdf

Thus at low pH values an amino acid exists as a cation and at high pH values as an

anion. At a particular intermediate pH the amino acid carries no net charge, although

it is still ionised, and is called a zwitterion. It has been shown that, in the crystalline

state and in solution in water, amino acids exist predominantly as this zwitterionic

form. This confers upon them physical properties characteristic of ionic compounds,

i.e. high melting point and boiling point, water solubility and low solubility in organic

solvents such as ether and chloroform. The pH at which the zwitterion predominates

in aqueous solution is referred to as the isoionic point, because it is the pH at which

the number of negative charges on the molecule produced by ionisation of the

carboxyl group is equal to the number of positive charges acquired by proton

acceptance by the amino group. In the case of amino acids this is equal to the

isoelectric point (pI), since the molecule carries no net charge and is therefore

electrophoretically immobile. The numerical value of this pH for a given amino acid

is related to its acid strength (pKavalues) by the equation:

pI¼pKa^1 þpKa^2

2

ð 8 : 1 Þ

where pKa1and pKa2are equal to the negative logarithm of the acid dissociation

constants,Ka1andKa2(Section 1.3.2).

In the case of glycine, pKa1and pKa2are 2, 3 and 9.6, respectively, so that the isoionic

point is 6.0. At pH values below this, the cation and zwitterion will coexist in equilib-

rium in a ratio determined by the Henderson–Hasselbalch equation (Section 1.3.3),

whereas at higher pH values the zwitterion and anion will coexist in equilibrium.

For acidic amino acids such as aspartic acid, the ionisation pattern is different

owing to the presence of a second carboxyl group:

COOH

2.1 3.9 9.8

COOH

Cation

(1 net

positive

charge)

Zwitterion

pH 3.0

(isoionic

point)

CH 2

CH

COOH

COO–

CH 2

CH

Anion

(1 net

negative

charge)

COO–

COO–

CH 2

CH

Anion

(2 net

negative

charges)

COO–

COO–

CH 2

NH 3 CH

+

NH 3

+

NH 3 NH 2

pKa 1 pKa 2 + pKa 3

In this case, the zwitterion will predominate in aqueous solution at a pH determined

by pKa 1 and pKa 2 , and the isoelectric point is the mean of pKa 1 and pKa 2.

Table 8.1 (cont.)

Amino acid Three-letter code One-letter code

Tyrosine Tyr Y

Valine Val V

302 Protein structure, purification, characterisation and function analysis