1.4 Proteins 59Table 1.28.pK values of protein side chains
Group pK Group pK
(25◦C) (25◦C)
α-Carboxyl- 3–4 Imidazolium- 4–8
β,γ-Carboxyl- 3–5 Hydroxy-
α-Ammonium- 7–8 (aromatic) 9–12
ε-Ammonium- 9–11 Thiol 8–11
Guanidinium- 12–13
of 6–6.5, while the pK of theβ-carboxyl group of
Asp^66 is 1.5–2, of Asp^52 is 3–4.6 and of Asp^101
is 4.2–4.7.
The total charge of a protein, which is the ab-
solute sum of all positive and negative charges,
is differentiated from the so-called net charge
which, depending on the pH, may be positive,
zero or negative. By definition the net charge is
zero and the total charge is maximal at the iso-
electric point. Lowering or raising the pH tends
to increase the net charge towards its maximum,
while the total charge always becomes less than
at the isolectric point.
Since proteins interact not only with protons but
also with other ions, there is a further differentia-
tion between an isoionic and an isoelectric point.
The isoionic point is defined as the pH of a pro-
tein solution at infinite dilution, with no other
ions present except for H+and HO−. Such a pro-
tein solution can be acquired by extensive dialy-
Fig. 1.33.pH-shift of isoionic serum albumin solutions by added salts. (according toEdsallandWymann, 1958)
sis (or, better, electrodialysis) against water. The
isoionic point is constant for a given substance
while the isoelectric point is variable depending
on the ions present and their concentration. In
the presence of salts, i. e. when binding of an-
ions is stronger than that of cations, the isoelec-
tric point is lower than the isoionic point. The
reverse is true when cationic binding is domi-
nant. Figure 1.33 shows the shift in pH of an
isoionic serum albumin solution after addition
of various salts. The shift in pH is consistently
positive, i. e. the protein binds more anions than
cations.
The titration curve ofβ-lactoglobulin at various
ionic strengths (Fig. 1.34) shows that the isoelec-
tric point of this protein, at pH 5.18, is independ-
ent of the salts present. The titration curves are,
however, steeper with increasing ionic strength,
which indicates greater suppression of the elec-
trostatic interaction between protein molecules.
At its isoelectric point a protein is the least sol-
uble and the most likely to precipitate (“isoelec-
tric precipitation”) and is at its maximal crystal-
lization capacity. The viscosity of solubilized pro-
teins and the swelling power of insoluble proteins
are at a minimum at the isoelectric point.
When the amino acid composition of a protein is
known, the isoelectric point can be estimated ac-
cording to the following formula:(1.93)