pI = (pKa₁ + pKa₂) / 2
The isoelectric point (pI) is the pH at which a molecule carries no net electrical charge. For simple amino acids with only two ionizable groups: pI = (pKa₁ + pKa₂)/2. For amino acids with ionizable side chains (Lys, Arg, His, Asp, Glu, Cys, Tyr), pI is the average of the two pKa values that bracket the zwitterionic form. At pH < pI, the molecule is positively charged; at pH > pI, it is negatively charged. The pI concept is fundamental to: protein electrophoresis (SDS-PAGE, IEF), ion exchange chromatography, protein precipitation (minimum solubility at pI), and understanding protein-surface interactions. Protein pI values typically range from 4 to 11 and can be estimated from amino acid composition.