What the isoelectric point is
Amino acids carry ionizable groups: an α-carboxyl group (−COOH), an α-amino group (−NH₃⁺) and, for some, an ionizable side chain. The charge of each group depends on the pH. The isoelectric point (pI) is the pH at which the molecule has no net charge: it exists mostly as a neutral zwitterion.
How to calculate it
The pI is the average of the two pKa values on either side of the neutral form:
- Neutral side chain: pI = (pKa₁ + pKa₂) ÷ 2
- Acidic side chain (Asp, Glu, Cys): pI = (pKa₁ + pKaR) ÷ 2
- Basic side chain (Lys, Arg, His): pI = (pKa₂ + pKaR) ÷ 2
Tyrosine’s phenol group has a pKa above the amino group, so its pI is computed like a neutral amino acid.
Examples
- Glycine: (2.34 + 9.60) ÷ 2 = 5.97
- Aspartic acid: (1.88 + 3.65) ÷ 2 = 2.77
- Lysine: (8.95 + 10.53) ÷ 2 = 9.74
- Histidine: (6.00 + 9.17) ÷ 2 = 7.59
Net charge at a given pH
For each group, the Henderson-Hasselbalch equation gives the fraction that is charged. The calculator adds them up:
- a basic group contributes +1 ÷ (1 + 10^(pH − pKa));
- an acidic group contributes −1 ÷ (1 + 10^(pKa − pH)).
Lysine at pH 7.4 carries a net charge of about +0.97: its α-amino group and side chain are almost fully protonated, while the carboxyl group is fully deprotonated.
Why it matters
- Electrophoresis: below its pI, a molecule is positive and moves toward the cathode; above its pI, it is negative and moves toward the anode. Isoelectric focusing separates proteins exactly by pI.
- Ion exchange chromatography: choose a buffer pH about one unit away from the pI so the protein binds to the column.
- Solubility: proteins are least soluble at their pI, which is used to precipitate them, for example casein from milk at pH 4.6.
Limits
The pKa values used here are standard textbook values at 25 °C for free amino acids. In a peptide or protein, the pKa of each group shifts with its environment, and only the terminal groups and side chains ionize. For proteins, specialized tools sum every ionizable residue.