Histidine pKa and Isoelectric Point: How to Calculate pI for Amino Acids

The isoelectric point (pI) is the pH at which an amino acid has no net charge. You find it by averaging the two pKa values that bracket the neutral (zwitterion or net-zero) form.

The rule

1. List the pKa values in order, lowest to highest.
2. Mark the charge of the species between each pair.
3. Average the two pKa values on either side of the net-zero species.

For an acidic amino acid, average the two lowest pKa values. For a basic one, average the two highest.

Histidine

Histidine has three ionizable groups: alpha-COOH (about 1.8), imidazole side chain (about 6.0) and alpha-NH₃⁺ (about 9.2).

• Net charge goes +2, +1, 0, -1 as pH rises.
• The neutral form lies between the imidazole and amine pKa values: pI = (6.0 + 9.2)/2 = about 7.6.
• Because the side chain pKa is close to physiological pH, histidine is the only amino acid that is partly protonated and partly not at pH 7.4. That makes it a good buffer and a common catalytic acid or base in enzyme active sites.

Charge versus pH

• pH below pI: net positive. The molecule migrates to the cathode in electrophoresis.
• pH above pI: net negative. It migrates to the anode.
• At pI: no net charge and lowest solubility, which is why proteins precipitate near their pI.

Common traps

• Averaging all pKa values. Only the two around the net-zero form.
• Calling histidine "basic" without checking the pKa. At pH 7.4 it is mostly neutral, though classed as basic.

One-minute recap

• pI = average of the pKa values on either side of the neutral form.
• Acidic: two lowest. Basic: two highest.
• Histidine side chain pKa about 6.0; pI about 7.6.