Isoelectric point

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Overview

The isoelectric point (pI) 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. They are affected by pH of their surrounding environment and can become more positively or negatively charged due to the loss or gain of protons (H⁺). A molecule's pI can affect its solubility at a certain pH. Such molecules have minimum solubility in water or salt solutions at the pH which corresponds to their pI and are often seen to 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 be separated according to their isoelectric point (overall charge) on a polyacrylamide gel using a technique called isoelectric focusing. This technique utilises a pH gradient to separate proteins. Isoelectric focusing is also the first step in preforming 2-D gel polyacrylamide gel electrophoresis.

For an amino acid with only one amine and one carboxyl group, the pI can be calculated from the pKas of this molecule.

<math> pI = {{pK_1 + pK_2} \over 2} </math>

For amino acids with more than two ionizable groups, such as lysine, the same formula is used, but this time the two pKa's used are those of the two groups that lose and gain a charge from the neutral form of the amino acid. Lysine has a single carboxylic pKa and two amine pKa values (one of which is on the R-group), so fully protonated lysine has a +2 net charge. To get a neutral charge, we must deprotonate the lysine twice , and therefore use the R-group and amine pKa values (found at List of standard amino acids).

<math> pI = {{9.06 + 10.54} \over 2} = 9.80 </math>

However, a more exact treatment of this requires advanced acid/base knowledge and calculations.

Proteins can be separated according to their isoelectric point in a process known as isoelectric focusing.

At a pH below the pI, proteins carry a net positive charge. Above the pI they carry a net negative charge. This has implications for running electrophoretic gels (see Agarose gel electrophoresis). 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.

Isoelectric Point of Ceramic Materials

The isoelectric points (IEP) of metal oxide ceramics are used extensively in material science in various aqueous processing steps (synthesis, modification, etc.). For these surfaces, present as colloids or larger particles in aqueous solution, the surface is 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-OH⁺ species predominate. Some approximate values of common ceramics are listed below (Haruta and Brunelle, except where noted). The exact value can vary widely, depending on material factors such as purity and phase as well as physical parameters such as temperature. In addition, precise measurement of isoelectric points is difficult and requires careful techniques, even with modern methods. Thus, many sources often cite differing values for isoelectric points of these materials.

Examples of isoelectric points for various materials

The following list gives the pH_25°C of isoelectric point at 25 °C for selected materials in water:

Note: The list is ordered by increasing pH values.

• tungsten(VI) oxide WO₃: 0.2-0.5
• antimony(V) oxide Sb₂O₅: <0.4 to 1.9
• vanadium(V) oxide (vanadia) V₂O₅: 1-2 (3 )
• silicon oxide (silica) SiO₂: 1.7-3.5
• silicon carbide (alpha) SiC: 2-3.5
• tantalum(V) oxide, Ta₂O₅: 2.7-3.0
• gas bubbles (regardless of the nature of the gas): about 3
• tin(IV) oxide SnO₂: 4-5.5 (7.3 )
• zirconium(IV) oxide (zirconia) ZrO₂: 4-11
• manganese(IV) oxide MnO₂: 4-5
• delta-MnO₂ 1.5, beta-MnO₂ 7.3
• titanium(IV) oxide (titania) (rutile or anatase) TiO₂: 3.9-8.2
• silicon nitride Si₃N₄: 6-7
• iron (II, III) oxide (magnetite) Fe₃O₄: 6.5-6.8
• gamma iron (III) oxide (maghemite) Fe₂O₃: 3.3-6.7
• cerium(IV) oxide (ceria) CeO₂: 6.7-8.6
• chromium(III) oxide (chromia) Cr₂O₃: 7 (6.2-8.1 )
• gamma aluminum oxide (gamma alumina) Al₂O₃: 7-8
• boehmite (gamma AlOOH) 7.2-8.2
• thallium(I) oxide Tl₂O: 8
• alpha iron (III) oxide (hematite) Fe₂O₃: 8.4-8.5
• alpha aluminum oxide (alpha alumina, corundum) Al₂O₃: 8-9
• silicon nitride Si₃N₄: 9
• yttrium(III) oxide (yttria) Y₂O₃: 7.15-8.95
• copper(II) oxide CuO: 9.5
• zinc oxide ZnO: 8.7-10.3
• lanthanum(III) oxide La₂O₃: 10
• nickel(II) oxide NiO: 10-11 (9.9-11.3 )
• lead(II) oxide PbO: 10.7-11.6
• magnesium oxide (magnesia) MgO: 12-13 (9.8-12.7 )

Mixed oxides may exhibit isoelectric point values that are intermediate to those of the corresponding pure oxides. For example, Jara et al. measured an IEP of 4.5 for a synthetically-prepared amorphous aluminosilicate (Al₂O₃-SiO₂). The researchers noted that 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 (see Lewis). Lewis also lists the IEP of barium titanate, BaTiO₃ as being between pH 5 and 6, while Vamvakaki et al. reported a value of 3, although these authors note that a wide range of values have been reported, a result of either residual barium carbonate on the surface or TiO₂-rich surfaces.

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 the 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 the 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 surface.

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 net neutral surface charge. The difference between the two, therefore, is quantity of charged sites at the point of net zero charge. Jolivet uses the intrinsic surface equilbrium constants, pK^- and pK⁺ to define the two conditions in terms of the relative number of charged sites:

<math> pK^- - pK^+ = \Delta pK = \log {\frac{\left[MOH\right]^2}{\left[MOH{_2^+}\right]\left[MO^-\right]}} </math>

For large ΔpK (>4 according to Jolivet), the predominate 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.

Further reading

• Nelson DL, Cox MM (2004). Lehninger Principles of Biochemistry. W. H. Freeman; 4th edition (Hardcover). ISBN 0-7167-4339-

External links

• EMBL WWW Gateway to Isoelectric Point Service — calculates the pI for an input amino acid sequence.
• Calculation of protein isoelectric point — free online and offline program to calculation pI and more theoretical information about this subject.

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