Amino Acid Ionization & pI Values

Amino Acids as Zwitterions

At physiological pH (about 7.4), free amino acids typically exist predominantly as zwitterions: the alpha-amino group is protonated (NH₃⁺) and the alpha-carboxyl group is deprotonated (COO^-). The net charge is not fixed; it depends on how the solution pH compares to each ionizable group's pKa. When pH is below a group's pKa, that group tends to be protonated; when pH is above its pKa, it tends to be deprotonated.

pKa Values of Common Amino Acids

Reported pKa values depend slightly on temperature, ionic strength, and measurement method; the table below lists typical values used in biochemistry courses.

The Titration Curve

Titration of a simple amino acid with strong base yields a sigmoidal curve with two buffering regions, one near each pKa. At pH = pKa, the conjugate acid and conjugate base of that group are present in a 1:1 ratio. The Henderson-Hasselbalch equation relates pH, pKa, and the ratio of species:

pH = pKa + log([A^-]/[HA])

Buffering capacity is greatest within about one pH unit of each pKa, where the slope of the titration curve is shallowest. Between the two pKa values of a simple amino acid, the dominant species is the zwitterion; the pH at which net charge is zero (the isoelectric point) lies between those two pKa values.

Calculating the Isoelectric Point (pI)

The isoelectric point (pI) is the pH at which the molecule has no net charge. Operationally, it is the average of the two pKa values that bracket the zwitterionic form when you order all ionizable groups by increasing pKa.

• Simple amino acids (no ionizable side chain): the zwitterion lies between the alpha-carboxyl and alpha-amino pKa values, so pI = (pKa1 + pKa2) / 2.
• Acidic side chains (Asp, Glu): the neutral form is bracketed by pKa1 (alpha-COOH) and pKaR (side-chain COOH), so pI = (pKa1 + pKaR) / 2.
• Basic side chains (Lys, Arg, His): the neutral form is bracketed by the alpha-amino pKa and the side-chain pKa, so pI = (pKa2 + pKaR) / 2.

Worked examples