The definition
The isoelectric point, pI, is the pH at which a protein carries no net electric charge. Below its pI a protein is positively charged, because its basic groups are protonated; above it the protein is negative, because its acidic groups have lost their protons.
Where the charge comes from
Seven side chains and the two chain ends change charge with pH. Each has a pKa, the pH at which it is half charged. Approximate values:
- Acidic (negative above their pKa): C-terminus (about 3.5), aspartate (3.9), glutamate (4.1), cysteine (8.3), tyrosine (10.1).
- Basic (positive below their pKa): histidine (6.0), N-terminus (about 8.0), lysine (10.5), arginine (12.5).
The exact pKa values vary between tables (EMBOSS, Lehninger, Bjellqvist), which is why different tools give slightly different pI values for the same sequence.
How it is calculated
For a trial pH, the charge of each group is worked out with the Henderson-Hasselbalch equation: a basic group contributes +1 ÷ (1 + 10pH − pKa) and an acidic group −1 ÷ (1 + 10pKa − pH). Adding these over the whole sequence gives the net charge at that pH. The pH is then adjusted, by bisection, until the net charge is zero. A protein with many lysines and arginines ends up with a high pI; one rich in aspartate and glutamate has a low pI.
What pI is used for
- Isoelectric focusing and 2D gels: a protein migrates in a pH gradient until it reaches its pI and stops.
- Ion exchange chromatography: at a pH below its pI a protein binds a cation exchanger; above its pI, an anion exchanger.
- Solubility: proteins are least soluble near their pI, which is used for precipitation and is why storage buffers are kept at least a pH unit away from it.
Calculate it
The Protein Isoelectric Point Calculator gives the pI and the net charge at any pH you choose, for one sequence or many. Protein Stats adds the amino acid composition, and Protein Molecular Weight the mass in daltons.