ListMoto - Isoelectric Point

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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 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 . 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

* 1.1 Examples

* 2 Isoelectric point
Isoelectric point
of peptides and proteins * 3 Ceramic materials * 4 Isoelectric point
Isoelectric point
versus point of zero charge * 5 See also * 6 References * 7 Further reading * 8 External links


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. p I = p K a 1 + p K a 2 2 {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 positive charge.


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 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.


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:


WO3 0.2-0.5 Ta2O5 2.7-3.0 δ-MnO2 1.5 Fe2O3 3.3-6.7 Fe2O3 8.4-8.5 ZnO 8.7-10.3

Sb2O5 p K + = p K = log 2 {displaystyle mathrm {p} K^{-}-mathrm {p} K^{+}=Delta mathrm {p} K=log {frac {left^{2}}{leftleft}}}

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.


* Isoionic point * QPNC-PAGE
* pK acid dissociation constant * Henderson-Hasselbach equation * isoelectric focusing


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Separation Techniques" (PDF). Reciprocal Meat Conference Proceedings. 36: 98–102. * ^ For derivation of this expression see acid dissociation constant * ^ Bjellqvist, B.; Hughes, G. J.; Pasquali, C.; Paquet, N.; Ravier, F.; Sanchez, J. C.; Frutiger, S.; Hochstrasser, D. (1993-10-01). "The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences". Electrophoresis. 14 (10): 1023–1031. ISSN 0173-0835 . PMID 8125050 .

* ^ Gauci, Sharon; van Breukelen, Bas; Lemeer, Simone M.; Krijgsveld, Jeroen; Heck, Albert J. R. (2008-12-01). "A versatile peptide pI calculator for phosphorylated and N-terminal acetylated peptides experimentally tested using peptide isoelectric focusing". Proteomics. 8 (23–24): 4898–4906. ISSN 1615-9861 . PMID 19003858 . doi :10.1002/pmic.200800295 . * ^ Gasteiger, Elisabeth; Gattiker, Alexandre; Hoogland, Christine; Ivanyi, Ivan; Appel, Ron D.; Bairoch, Amos (2003-07-01). "ExPASy: the proteomics server for in-depth protein knowledge and analysis" . Nucleic Acids Research. 31 (13): 3784–3788. ISSN 0305-1048 . PMC 168970  . PMID 12824418 . doi :10.1093/nar/gkg563 . * ^ Cargile, Benjamin J.; Sevinsky, Joel R.; Essader, Amal S.; Eu, Jerry P.; Stephenson, James L. (2008-07-01). "Calculation of the isoelectric point of tryptic peptides in the pH 3.5-4.5 range based on adjacent amino acid effects". Electrophoresis. 29 (13): 2768–2778. ISSN 0173-0835 . PMID 18615785 . doi :10.1002/elps.200700701 . * ^ Perez-Riverol, Yasset; Audain, Enrique; Millan, Aleli; Ramos, Yassel; Sanchez, Aniel; Vizcaíno, Juan Antonio; Wang, Rui; Müller, Markus; Machado, Yoan J. (2012-04-03). " Isoelectric point
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* Nelson DL, Cox MM (2004). Lehninger Principles of Biochemistry. W. H. Freeman; 4th edition (Hardcover). ISBN 0-7167-4339-6 * Kosmulski M. (2009). Surface Charging and Points of Zero Charge. CRC Press; 1st edition (Hardcover). ISBN 978-1-4200-5188-9


* IPC – Isoelectric Point