What is the Isoelectric Point?

Proteins are built of chains of amino acids, each of which have different pH values. The overall pH of the protein is composed of the mixture of the pH values of the individual amino acids as they form ions in the particular solution in which they are dissolved. The isoelectric point (pI) of a protein is the pH at which that protein has no net charge. This property can be exploited to separate the protein with the known pI from other proteins in a heterogeneous mixture.

Amino acids have an amino terminal group that is basic, having a high pH. The other end of the amino acid is the carboxyl terminal that is acidic, with a low pH. At differing pH values, the amino acids on the proteins will vary in their charges. Proteins below their isoelectric point have a positive charge. In contrast, those above this point have a negative charge.

To exploit knowledge of the isoelectric point for protein purification, a mixture of proteins is subjected to an electric field. This is commonly done in agarose or polyacrylamide gels, and is known as isoelectric focusing. An older technique is to perform the procedure on a larger scale in a glass column using a solution of sucrose with electrodes on each end. Compounds called ampholytes are added that cause the formation of a consistent pH gradient. When the gel or column is subjected to the electric current, the proteins migrate until they reach their isoelectric point, and then remain stationary.

Proteins on gels are generally made visible by a dye that binds proteins. Sometimes, if enzymes are being studied, a substrate can be used that gives a colored reaction. Usually standards are used that have proteins of known isoelectric points.

Once one knows where the desired protein is located, a common technique is to cut the isolated protein out of the gel. The protein can then be purified and sequenced. Once the sequence is known, it can be used to design primers for the polymerase chain reaction (pcr) and used to clone the gene for the protein if suitable nucleic acid material is available.

Isoelectric focusing is also a common way to analyze closely related proteins to see how different they are from each other. One complication can be that proteins can have sugars bound to them. This is called glycosylation and can affect the protein’s pI. It may look like there are multiple proteins with different isoelectric points, when in fact there is just one protein that has been differentially glycosylated. Proteins purified by standard methods such as chromatography are sometimes analyzed by isoelectric focusing to ensure their purity.