Download basic principles of isoelectric focusing in biomedical engineering

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2013
BASIC PRINCIPLES OF ISOELECTRIC FOCUSING IN BIOMEDICAL
ENGINEERING
Dr.h.c. prof. Ing. Jozef Živčák, PhD.
Department of Biomedical Engineering and
Measurement DBEaM, TU
Letná 9, 042 00 Kosice, Slovakia
e-mail: [email protected]
RNDr. Marianna Trebuňová, PhD.
Associated Tissue Bank
Rastislavova 43, Kosice, Slovakia
e-mail: [email protected]
Abstract
Isoelectric focusing (IEF) is
an
electrophoretic technique for the separation of
proteins based on their isoelectric point (pI). The pI
is the pH at which a protein has no net charge and
thus, does not migrate further in an electric field.
IEF gels are used to determine the isoelectric point
(pI) of a protein and to detect minor changes in the
protein due to post-translational modifications such
as phosphorylation and glycosylation [1].
Key words: Isoelectric focusing, isoelectric point,
proteins
electrical charge or the negative and positive
charges are equal.
Surfaces naturally charge to form a double
layer. In the common case when the surface chargedetermining ions are H+/OH-, the net surface charge
is affected by the pH of the liquid in which the solid
is submerged. Again, the pI is the pH value of the
solution at which the surfaces carries no net charge.
The pI value can affect the solubility of a
molecule at a given pH. Such molecules have
minimum solubility in water or salt solutions at the
pH which corresponds to their pI and often
precipitate out of solution. Biological amphoteric
molecules such as proteins contain both acidic and
basic functional groups. Amino acids which make
up proteins may be positive, negative, neutral or
polar in nature, and together give a protein its
overall charge. At a pH below their pI, proteins
carry a net positive charge; above their pI they
carry a net negative charge. Proteins can thus be
separated according to their isoelectric point
(overall charge) on a polyacrylamide gel using a
technique called isoelectric focusing, which uses a
pH gradient to separate proteins. Isoelectric
focusing is also the first step in 2-D gel
polyacrylamide gel electrophoresis [2].
In t ro du ct io n
In isoelectric focusing (IEF), proteins are
applied to polyacrylamide gels (IEF gels) or
immobilized pH gradient (IPG) strips containing a
fixed pH gradient. An electrical field is applied and
the protein sample containing a mixture of proteins
migrates through the pH gradient. Individual
proteins are immobilized in the pH gradient as they
approach their specific pI. After staining the gel and
documenting the results, proteins separated by pI
can be separated by mass using 2D gel
electrophoresis [1].
Isoelectric point
The isoelectric point (pI), sometimes
abbreviated to IEP, is the pH at which a particular
molecule or surface carries no net electrical charge.
Amphoteric molecules called zwitterions
contain both positive and negative charges
depending on the functional groups present in the
molecule. The net charge on the molecule is
affected by pH of their surrounding environment
and can become more positively or negatively
charged due to the loss or gain of protons (H+). The
pI is the pH value at which the molecule carries no
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Theoretical aspects
When a protein is at the position of its isoelectric
point, it has no net charge and cannot be moved in a
gel matrix by the electric field. It may, however,
move from that position by diffusion. The pH
gradient forces a protein to remain in its isoelectric
point position, thus concentrating it; this
concentrating effect is called "focusing". Increasing
the applied voltage or reducing the sample load
result in improved separation of bands. The applied
voltage is limited by the heat generated, which must
be dissipated. The use of thin gels and an efficient
cooling plate controlled by a thermostatic circulator
prevents the burning of the gel whilst allowing
sharp focusing. The separation is estimated by
determining the minimum pI difference (ΔpI),
which is necessary to separate 2 neighbouring
bands:
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D = diffusion coefficient of the protein,
•
for positive charged macromolecules:
= pH gradient,
E = intensity of the electric field, in volts per
centimetre,
= variation of the solute mobility with the pH
in the region close to the pI.
Since D and
for a given protein
cannot be altered, the separation can be improved
by using a narrower pH range and by increasing the
intensity of the electric field.
Resolution between protein bands on an
IEF gel prepared with carrier ampholytes can be
quite good. Improvements in resolution may be
achieved by using immobilised pH gradients where
the buffering species, which are analogous to
carrier ampholytes, are copolymerised within the
gel matrix. Proteins exhibiting pIs differing by as
little as 0.02 pH units may be resolved using a gel
prepared
with
carrier
ampholytes
while
immobilised pH gradients can resolve proteins
differing by approximately 0.001 pH units [3].
How to calculate isoelectric point of protein
where pKp is the acid dissociation constant
of positively charged amino acid.
We 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 [4].
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 (Picture 1) [4].
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 (NH2 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,
Picture 1 Scheme of Isoelectric focusing Source:
[5].
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CONCLUSION
IEF is one of the principal techniques
available for analyzing and characterizing proteins,
peptides, glyco- or lipoproteins, cell membranes
proteins, isoenzymes, etc. It can be very useful in
studying factors that influence the net molecular
charge of a molecule or a complex between
macromolecules. It permits us to analyze ligand
binding to proteins and to investigate the subunit
structures in multimeric proteins [6].
2013
[3] http://lib.njutcm.edu.cn/yaodian/ep/EP5.0/02_
methods_of_analysis/2.2.__physical_and_physicoc
hemical_methods/2.2.54.%20Isoelectric%20focusin
g.pdf
[4] http://isoelectric.ovh.org/files/isoelectric-pointtheory.html.
[5] http://en.wikipedia.org/wiki/File:Isoelectric_fo
cusing_contribute2.jpg
[6] Prats, M.: Minireview: Isoelectric Focusing in
Experimental sectiojn, Biochemical education, 20
(2), 1992, p. 109 – 111
References
[1] http://www.lifetechnologies.com/sk/en/home/li
fe-science/protein-expression-and-analysis/proteingel-electrophoresis/protein-gels/specialized-proteinseparation/isoelectric-focusing.html
[2] http://www.princeton.edu/~achaney/tmve/wiki
100k/docs/Isoelectric_point.html
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Acknowledgement
This study was supported by the grant
VEGA 1/0515/13 of the Ministry of Education of
the Slovak Republic and by the Agency of the
Slovak Ministry of Education.