The isoelectric point (pI, pH(I), IEP), is the pH at which a
particular molecule carries no net electrical charge in the
statistical mean. The standard nomenclature to represent the
isoelectric point is pH(I), although pI is also commonly seen,
and is used in this article for brevity. The net charge on the
molecule is affected by pH of its surrounding environment and can
become more positively or negatively charged due to the gain or loss,
respectively, of protons (H+).
Surfaces naturally charge to form a double layer. In the common case
when the surface charge-determining ions are H+/OH−, the net surface
charge is affected by the pH of the liquid in which the solid is
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 that 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 that 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 by net charge in a
polyacrylamide gel using either preparative gel electrophoresis, which
uses a constant pH to separate proteins or isoelectric focusing, which
uses a pH gradient to separate proteins.
Isoelectric focusing is also
the first step in 2-D gel polyacrylamide gel electrophoresis.
In biomolecules, proteins can be separated by ion exchange
chromatography. Biological proteins are made up of zwitterionic amino
acid compounds; the net charge of these proteins can be positive or
negative depending on the pH of the environment. The specific pI of
the target protein can be used to model the process around and the
compound can then be purified from the rest of the mixture. Buffers of
various pH can be used for this purification process to change the pH
of the environment. When a mixture containing a target protein is
loaded into an ion exchanger, the stationary matrix can be either
positively-charged (for mobile anions) or negatively-charged (for
mobile cations). At low pH values, the net charge of most proteins in
the mixture is positive - in cation exchangers, these
positively-charged proteins bind to the negatively-charged matrix. At
high pH values, the net charge of most proteins is negative, where
they bind to the positively-charged matrix in anion exchangers. When
the environment is at a pH value equal to the protein's pI, the net
charge is zero, and the protein is not bound to any exchanger, and
therefore, can be eluted out.
1 Calculating pI values
Isoelectric point of peptides and proteins
3 Ceramic materials
Isoelectric point versus point of zero charge
5 See also
7 Further reading
8 External links
Calculating pI values
For an amino acid with only one amine and one carboxyl group, the pI
can be calculated from the mean of the pKas of this molecule.
displaystyle mathrm pI = frac mathrm p K_ mathrm a1
+mathrm p K_ mathrm a2 2
The pH of an electrophoretic gel is determined by the buffer used for
that gel. If the pH of the buffer is above the pI of the protein being
run, the protein will migrate to the positive pole (negative charge is
attracted to a positive pole). If the pH of the buffer is below the pI
of the protein being run, the protein will migrate to the negative
pole of the gel (positive charge is attracted to the negative pole).
If the protein is run with a buffer pH that is equal to the pI, it
will not migrate at all. This is also true for individual amino acids.
glycine pK = 2.72, 9.60
adenosine monophosphate pK = 0.9, 3.8, 6.1
In the two examples (on the right) the isoelectric point is shown by
the green vertical line. In glycine the pK values are separated by
nearly 7 units so the concentration of the neutral species, glycine
(GlyH), is effectively 100% of the analytical glycine concentration.
Glycine may exist as a zwitterion at the isoelectric point, but the
equilibrium constant for the isomerization reaction in solution
H2NCH2CO2H ⇌ H3N+CH2CO2−
is not known.
The other example, adenosine monophosphate is shown to illustrate the
fact that a third species may, in principle, be involved. In fact the
concentration of (AMP)H32+ is negligible at the isoelectric point in
this case. If the pI is greater than the pH, the molecule will have a
Isoelectric point of peptides and proteins
A number of algorithms for estimating isoelectric points of peptides
and proteins have been developed. Most of them use
Henderson–Hasselbalch equation with different pK values. For
instance, within the model proposed by Bjellqvist and co-workers the
pK's were determined between closely related immobilines, by focusing
the same sample in overlapping pH gradients. Some improvements in
the methodology (especially in the determination of the pK values for
modified amino acids) have been also proposed. More advanced
methods take into account the effect of adjacent amino acids ±3
residues away from a charged aspartic or glutamic acid, the effects on
free C terminus, as well as they apply a correction term to the
corresponding pK values using genetic algorithm. Other recent
approaches are based on a support vector machine algorithm and pKa
optimization against experimentally known protein/peptide isoelectric
Moreover, experimentally measured isoelectric point of proteins were
aggregated into the databases. Recently, a database of
isoelectric points for all proteins predicted using most of the
available methods had been also developed.
The isoelectric points (IEP) of metal oxide ceramics are used
extensively in material science in various aqueous processing steps
(synthesis, modification, etc.). In the absence of chemisorbed or
physisorbed species particle surfaces in aqueous suspension are
generally assumed to be covered with surface hydroxyl species, M-OH
(where M is a metal such as Al, Si, etc.). At pH values above the
IEP, the predominate surface species is M-O−, while at pH values
below the IEP, M-OH2+ species predominate. Some approximate values of
common ceramics are listed below:
Note: The following list gives the isoelectric point at 25 °C
for selected materials in water. The exact value can vary widely,
depending on material factors such as purity and phase as well as
physical parameters such as temperature. Moreover, the precise
measurement of isoelectric points can be difficult, thus many sources
often cite differing values for isoelectric points of these materials.
Mixed oxides may exhibit isoelectric point values that are
intermediate to those of the corresponding pure oxides. For example, a
synthetically prepared amorphous aluminosilicate (Al2O3-SiO2) was
initially measured as having IEP of 4.5 (the electrokinetic behavior
of the surface was dominated by surface Si-OH species, thus explaining
the relatively low IEP value). Significantly higher IEP values (pH
6 to 8) have been reported for 3Al2O3-2SiO2 by others. Similarly,
also IEP of barium titanate, BaTiO3 was reported in the range 5-6
while others got a value of 3.
Isoelectric point versus point of zero charge
The terms isoelectric point (IEP) and point of zero charge (PZC) are
often used interchangeably, although under certain circumstances, it
may be productive to make the distinction.
In systems in which H+/OH− are the interface potential-determining
ions, the point of zero charge is given in terms of pH. The pH at
which the surface exhibits a neutral net electrical charge is the
point of zero charge at the surface. Electrokinetic phenomena
generally measure zeta potential, and a zero zeta potential is
interpreted as the point of zero net charge at the shear plane. This
is termed the isoelectric point. Thus, the isoelectric point is
the value of pH at which the colloidal particle remains stationary in
an electrical field. The isoelectric point is expected to be somewhat
different than the point of zero charge at the particle surface, but
this difference is often ignored in practice for so-called pristine
surfaces, i.e., surfaces with no specifically adsorbed positive or
negative charges. In this context, specific adsorption is
understood as adsorption occurring in a Stern layer or chemisorption.
Thus, point of zero charge at the surface is taken as equal to
isoelectric point in the absence of specific adsorption on that
According to Jolivet, in the absence of positive or negative
charges, the surface is best described by the point of zero charge. If
positive and negative charges are both present in equal amounts, then
this is the isoelectric point. Thus, the PZC refers to the absence of
any type of surface charge, while the IEP refers to a state of neutral
net surface charge. The difference between the two, therefore, is the
quantity of charged sites at the point of net zero charge. Jolivet
uses the intrinsic surface equilibrium constants, pK− and pK+ to
define the two conditions in terms of the relative number of charged
displaystyle mathrm p K^ - -mathrm p K^ + =Delta mathrm p
K=log frac left[mathrm MOH right]^ 2 left[mathrm MOH _ 2 ^ +
right]left[mathrm MO ^ - right]
For large ΔpK (>4 according to Jolivet), the predominant species
is MOH while there are relatively few charged species - so the PZC is
relevant. For small values of ΔpK, there are many charged species in
approximately equal numbers, so one speaks of the IEP.
pK acid dissociation constant
^ Acceptable variants on pH(I) would include pHI, pHIEP, etc; the main
point is that one cannot take the 'power' of I, rather one measures
the pH subject to a nominated condition.
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IPC – Isoelectric Point Calculator — calculate protein isoelectric
point using over 15 methods
prot pi - protein isoelectric point — an online program for
calculating pI of proteins (include multiple subunits and
CurTiPot — a suite of spreadsheets for computing acid-base
equilibria (charge versus pH plot of amphoteric molecules e.g., amino
SWISS-2DPAGE — a database of isoelectric points coming from
two-dimensional polyacrylamide gel electrophoresis (~ 2,000 proteins)
PIP-DB — a
Protein Isoelectric Point database (~ 5,000 proteins)
Proteome-pI — a proteome isoelectric point database (predicted