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The isoelectric point (pI, pH(I), IEP), is the pH at which a molecule carries no net
electrical charge Electricity is the set of physical phenomena associated with the presence and motion of matter that has a property of electric charge. Electricity is related to magnetism, both being part of the phenomenon of electromagnetism, as described by ...
or is electrically neutral in the
statistical mean In mathematics and statistics, the arithmetic mean ( ) or arithmetic average, or just the ''mean'' or the ''average'' (when the context is clear), is the sum of a collection of numbers divided by the count of numbers in the collection. The colle ...
. The standard nomenclature to represent the isoelectric point is pH(I). However, pI is also used. For brevity, this article uses pI. 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+/HO, the net surface charge is affected by the pH of the liquid in which the solid is submerged. 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 Solution may refer to: * Solution (chemistry), a mixture where one substance is dissolved in another * Solution (equation), in mathematics ** Numerical solution, in numerical analysis, approximate solutions within specified error bounds * Solutio ...
. Biological
amphoteric In chemistry, an amphoteric compound () is a molecule or ion that can react both as an acid and as a base. What exactly this can mean depends on which definitions of acids and bases are being used. One type of amphoteric species are amphiprot ...
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 Polyacrylamide gel electrophoresis (PAGE) is a technique widely used in biochemistry, forensic chemistry, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their ...
using either preparative gel electrophoresis, which uses a constant pH to separate proteins or
isoelectric focusing Isoelectric focusing (IEF), also known as electrofocusing, is a technique for separating different molecules by differences in their isoelectric point (pI). It is a type of zone electrophoresis usually performed on proteins in a gel that takes a ...
, 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 Ion chromatography (or ion-exchange chromatography) separates ions and polar molecules based on their affinity to the ion exchanger. It works on almost any kind of charged molecule—including large proteins, small nucleotides, and amino acid ...
. 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.


Calculating pI values

For an
amino acid Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha am ...
with only one
amine In chemistry, amines (, ) are compounds and functional groups that contain a basic nitrogen atom with a lone pair. Amines are formally derivatives of ammonia (), wherein one or more hydrogen atoms have been replaced by a substituent suc ...
and one
carboxyl In organic chemistry, a carboxylic acid is an organic acid that contains a carboxyl group () attached to an R-group. The general formula of a carboxylic acid is or , with R referring to the alkyl, alkenyl, aryl, or other group. Carboxyli ...
group, the pI can be calculated from the mean of the
pKa PKA may refer to: * Professionally known as: ** Pen name ** Stage persona * p''K''a, the symbol for the acid dissociation constant at logarithmic scale * Protein kinase A, a class of cAMP-dependent enzymes * Pi Kappa Alpha, the North-American so ...
s of this molecule. : \mathrm = \frac The pH of an electrophoretic gel is determined by the
buffer Buffer may refer to: Science * Buffer gas, an inert or nonflammable gas * Buffer solution, a solution used to prevent changes in pH * Buffering agent, the weak acid or base in a buffer solution * Lysis buffer, in cell biology * Metal ion 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.


Examples

In the two examples (on the right) the isoelectric point is shown by the green vertical line. In
glycine Glycine (symbol Gly or G; ) is an amino acid that has a single hydrogen atom as its side chain. It is the simplest stable amino acid ( carbamic acid is unstable), with the chemical formula NH2‐ CH2‐ COOH. Glycine is one of the proteinogen ...
the pK values are separated by nearly 7 units. Thus in the gas phase, 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 Adenosine monophosphate (AMP), also known as 5'-adenylic acid, is a nucleotide. AMP consists of a phosphate group, the sugar ribose, and the nucleobase adenine; it is an ester of phosphoric acid and the nucleoside adenosine. As a substituent it t ...
is shown to illustrate the fact that a third species may, in principle, be involved. In fact the concentration of is negligible at the isoelectric point in this case. If the pI is greater than the pH, the molecule will have a positive charge.


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 Glutamic acid (symbol Glu or E; the ionic form is known as glutamate) is an α-amino acid that is used by almost all living beings in the biosynthesis of proteins. It is a non-essential nutrient for humans, meaning that the human body can syn ...
, the effects on free C terminus, as well as they apply a correction term to the corresponding pK values using
genetic algorithm In computer science and operations research, a genetic algorithm (GA) is a metaheuristic inspired by the process of natural selection that belongs to the larger class of evolutionary algorithms (EA). Genetic algorithms are commonly used to gene ...
. Other recent approaches are based on a support vector machine algorithm and pKa optimization against experimentally known protein/peptide isoelectric points. 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. In practice, a protein with an excess of basic aminoacids (arginine, lysine and/or histidine) will bear an isoelectric point roughly greater than 7 (basic), while a protein with an excess of acidic aminoacids (aspartic acid and/or glutamic acid) will often have an isoelectric point lower than 7 (acidic). The electrophoretic linear (horizontal) separation of proteins by Ip along a pH gradient in a polyacrylamide gel (also known as
isoelectric focusing Isoelectric focusing (IEF), also known as electrofocusing, is a technique for separating different molecules by differences in their isoelectric point (pI). It is a type of zone electrophoresis usually performed on proteins in a gel that takes a ...
), followed by a standard molecular weight linear (vertical) separation in a second polyacrylamide gel ( SDS-PAGE), constitutes the so called
two-dimensional gel electrophoresis Two-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins. Mixtures of proteins are separated by two properties in two dimensions on 2D gels. 2-DE was first ...
or PAGE 2D. This technique allows a thorough separation of proteins as distinct "spots", with proteins of high molecular weight and low Ip migrating to the upper-left part of the bidimensional gel, while proteins with low molecular weight and high Ip locate to the bottom-right region of the same gel.


Ceramic materials

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 predominant 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 Aluminosilicate minerals ( IMA symbol: Als) are minerals composed of aluminium, silicon, and oxygen, plus countercations. They are a major component of kaolin and other clay minerals. Andalusite, kyanite, and sillimanite are naturally occurri ...
(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 Barium titanate (BTO) is an inorganic compound with chemical formula BaTiO3. Barium titanate appears white as a powder and is transparent when prepared as large crystals. It is a ferroelectric, pyroelectric, and piezoelectric ceramic material ...
, BaTiO3 was reported in the range 5-6 while others got a value of 3. Mixtures of
titania Titania may refer to: Astronomy * Titania (moon), the largest moon of the planet Uranus * 593 Titania, an asteroid Chemistry and mineralogy * Titania, an alternate name for titanium dioxide Fiction * Titania (A Midsummer Night's Dream), the Quee ...
(TiO2) and
zirconia Zirconium dioxide (), sometimes known as zirconia (not to be confused with zircon), is a white crystalline oxide of zirconium. Its most naturally occurring form, with a monoclinic crystalline structure, is the mineral baddeleyite. A dopant stabi ...
(ZrO2) were studied and found to have an isoelectric point between 5.3-6.9, varying non-linearly with %(ZrO2). The surface charge of the mixed oxides was correlated with acidity. Greater titania content led to increased Lewis acidity, whereas zirconia-rich oxides displayed Br::onsted acidity. The different types of acidities produced differences in ion adsorption rates and capacities.


Isoelectric point versus point of zero charge

The terms isoelectric point (IEP) and
point of zero charge The point of zero charge (pzc) is generally described as the pH at which the net charge of total particle surface (i.e. absorbent's surface) is equal to zero, which concept has been introduced in the studies dealt with colloidal flocculation to ex ...
(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 Zeta potential is the electrical potential at the slipping plane. This plane is the interface which separates mobile fluid from fluid that remains attached to the surface. Zeta potential is a scientific term for electrokinetic potential in coll ...
, and a zero zeta potential is interpreted as the point of zero net charge at the shear plane. This is termed the isoelectric point.A.W. Adamson, A.P. Gast, "Physical Chemistry of Surfaces", John Wiley and Sons, 1997. 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 from 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 Chemisorption is a kind of adsorption which involves a chemical reaction between the surface and the adsorbate. New chemical bonds are generated at the adsorbent surface. Examples include macroscopic phenomena that can be very obvious, like cor ...
. Thus, point of zero charge at the surface is taken as equal to isoelectric point in the absence of specific adsorption on that surface. According to Jolivet,Jolivet J.P., ''Metal Oxide Chemistry and Synthesis. From Solution to Solid State'', John Wiley & Sons Ltd. 2000, (English translation of the original French text, ''De la Solution à l'Oxyde'', InterEditions et CNRS Editions, Paris, 1994). 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, p''K'' and p''K''+ to define the two conditions in terms of the relative number of charged sites: : \mathrmK^- - \mathrmK^+ = \Delta \mathrmK = \log For large Δp''K'' (>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 Δp''K'', there are many charged species in approximately equal numbers, so one speaks of the IEP.


See also

* Henderson-Hasselbalch equation *
Isoelectric focusing Isoelectric focusing (IEF), also known as electrofocusing, is a technique for separating different molecules by differences in their isoelectric point (pI). It is a type of zone electrophoresis usually performed on proteins in a gel that takes a ...
*
Zeta potential Zeta potential is the electrical potential at the slipping plane. This plane is the interface which separates mobile fluid from fluid that remains attached to the surface. Zeta potential is a scientific term for electrokinetic potential in coll ...
* Electrophoretic deposition * Isoionic point * pK acid dissociation constant *
QPNC-PAGE QPNC-PAGE, or quantitative preparative native continuous polyacrylamide gel electrophoresis, is a bioanalytical, high-resolution and highly accurate technique applied in biochemistry and bioinorganic chemistry to separate proteins quantitatively ...


References


Further reading

* Nelson DL, Cox MM (2004). ''Lehninger Principles of Biochemistry''. W. H. Freeman; 4th edition (Hardcover). * Kosmulski M. (2009). ''Surface Charging and Points of Zero Charge''. CRC Press; 1st edition (Hardcover).


External links


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 posttranslational modifications)

— a suite of spreadsheets for computing acid-base equilibria (charge versus pH plot of amphoteric molecules e.g., amino acids)
pICalculax
— Isoelectric point (pI) predictor for chemically modified peptides and proteins
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 isoelectric point for all proteins) {{DEFAULTSORT:Isoelectric Point Ions Molecular biology