The Isoelectric Point

As defined above, the isoelectric point, pI, is the pH of an aqueous solution of an amino acid (or peptide) at which the molecules on average have no net charge. In other words, the positively charged groups are exactly balanced by the negatively charged groups. For simple amino acids such as alanine, the pI is an average of the pK_a's of the carboxyl (2.34) and ammonium (9.69) groups. Thus, the pI for alanine is calculated to be: (2.34 + 9.69)/2 = 6.02, the experimentally determined value. If additional acidic or basic groups are present as side-chain functions, the pI is the average of the pK_a's of the two most similar acids. To assist in determining similarity we define two classes of acids. The first consists of acids that are neutral in their protonated form (e.g. CO₂H & SH). The second includes acids that are positively charged in their protonated state (e.g. -NH₃⁺). In the case of aspartic acid, the similar acids are the alpha-carboxyl function (pK_a = 2.1) and the side-chain carboxyl function (pK_a = 3.9), so pI = (2.1 + 3.9)/2 = 3.0. For arginine, the similar acids are the guanidinium species on the side-chain (pK_a = 12.5) and the alpha-ammonium function (pK_a = 9.0), so the calculated pI = (12.5 + 9.0)/2 = 10.75.