Polish version |
|
Theoretical basis of isoelectric
point calculation, i.e. how to calculate isoelectric point of protein
Let's start from isoelectric point definition:
Isoelectric
point (pI) is
a pH in which net charge of protein is zero. In case of proteins
isoelectric point
mostly depends on seven charged amino acids: glutamate
(δ-carboxyl group), aspartate (ß-carboxyl
group),
cysteine (thiol group), tyrosine (phenol group),
histidine (imidazole side chains), lysine (
ε-ammonium group)
and
arginine (guanidinium group). Additonally, one should take into account
charge of protein terminal groups (NH
2
i COOH). Each of them has its unique acid dissociation constant
referred to as
pK.
Moreover, net charge of the protein is in tight relation with the
solution (buffer) pH. Keeping in main this we can use
Henderson-Hasselbach
equation to calculate protein charge in certain pH:
- for negative charged
macromolecules:
where pKn
is the acid dissociation constant of negatively charged amino acid
where pK
p is
the acid dissociation constant of positively charged amino acid
As you can see,
only pH of buffer is variable
in equations. If we successively change this value,
finally
we will find isoelectric point of analyzed protein. The knowledge
of isoelectric point
is
of great significance in biochemistry (mainly in elecrophoresis and
isofocusing techniques), because it allows to match proper environment
before the experiment starts.
Generally, macromolecules
are positively charged and on the other hand, above proteins isoelectric
point, their
charge
is negative.For example, during electrophoresis, direction of proteins migration, depends only
from their charge. If buffer pH (and as a result gel pH)
is higher
than protein isoelectric point, the particles will migrate to
the anode (negative
electrode) and if the buffer pH is lower
than isoelectric point
they will go to the cathode. In
situation when the gel pH and the protein isoelectric
point are
equal, proteins do
not move at all.
Using above formulae, we can calculate theoretical isoelectric point.
The result will be almost surely different than
real isoelectric point. It is mainly
because many proteins are chemically modified (amino acids can be
phosphorylated, methylated, acetyleted etc.), which change their
charge.
Problematic is also the occurrence of cysteines (negative
charge) which
can oxidise and form disulfide bond in
protein. Therefore,
they will become cystines, which do not express any charge.
Nevertheless, one can
approximately calculate protein isoelectric point which
is ± 0.5 of exact isoelectric point. The
most critical
moment
during isoelectric point determination is usage of appropriate pK
values. Unfortunately, there is no agreement in this matter. Each
source gives different pKs. Some of them are presented below:
|
Amino acid |
NH2 |
COOH |
C |
D |
E |
H |
K |
R |
Y |
| EMBOSS |
8.6 |
3.6 |
8.5 |
3.9 |
4.1 |
6.5 |
10.8 |
12.5 |
10.1 |
| DTASelect |
8.0 |
3.1 |
8.5 |
4.4 |
4.4 |
6.5 |
10.0 |
12.0 |
10.0 |
| Solomon |
9.6 |
2.4 |
8.3 |
3.9 |
4.3 |
6.0 |
10.5 |
12.5 |
10.1 |
| Sillero |
8.2 |
3.2 |
9.0 |
4.0 |
4.5 |
6.4 |
10.4 |
12.0 |
10.0 |
| Rodwell |
8.0 |
3.1 |
8.33 |
3.68 |
4.25 |
6.0 |
11.5 |
11.5 |
10.07 |
| Patrickios |
11.2 |
4.2 |
- |
4.2 |
4.2 |
- |
11.2 |
11.2 |
- |
| Wikipedia |
8.2 |
3.65 |
8.18 |
3.9 |
4.07 |
6.04 |
10.54 |
12.48 |
10.46 |
Now, having this few peaces of information we can try to
write
simple computer program which calculate isoelectric point. We will use
free
compiler
DevC++
as the program will be written in C++ programming language. To read
next section
you should have at least basic knowledge in C++.
For more theoretical information go to:
http://en.wikipedia.org/wiki/Isoelectric_point
Tabb DL (2001) An algorithm for isoelectric point estimation
Sillero A, Maldonado A. (2006) Isoelectric point determination of
proteins and other macromolecules: oscillating method. Comput Biol Med.
36(2), 157-66. Epub 2005 Jan 1 - this one is not open access article