What is a zwitterion?

A zwitterion is a molecule that carries both a positive and a negative charge at the same time, with the two canceling to a net charge of zero. Amino acids at their isoelectric point are zwitterions: the amino group is protonated (positive) while the carboxyl group is deprotonated (negative).

For an amino acid with 3 pKa values, why isn't pI the average of all three?

Only the two pKa values that directly flank the neutral, net-zero species determine the pI, a third pKa far outside that range (already fully protonated or fully deprotonated at the relevant pH) doesn't affect where the charge crosses zero. Averaging all three pKa values regardless of which ones actually flank the neutral point is a common exam mistake.

Why do basic amino acids have a pI above 7 and acidic ones below 7?

An amino acid needs extra base (higher pH) to strip the last proton and reach net-zero charge when it has an additional protonatable (basic) group, which is why lysine and arginine have high pIs. Conversely, an extra acidic side chain group makes the molecule net-negative until a lower pH, pulling the pI down, which is why aspartate and glutamate have low pIs.

Why does histidine's side chain pKa matter so much in biochemistry?

Histidine's side-chain pKa (~6.0) sits close to physiological pH (7.4), so it's one of the few side chains that's meaningfully both protonated and deprotonated near neutral pH. That makes it especially useful as a proton donor/acceptor in enzyme active sites and as a natural buffering component in proteins.

Glutamic Acid Titration Curve: pKa 2.19, 4.25, 9.67, pI 3.22

Glutamic acid has 3 ionizable groups, with pKa values of 2.19, 4.25 and 9.67. Its isoelectric point is pI 3.22, and the curve has 2 equivalence points and 3 buffering plateaus.

The ionizable groups

Group	pKa	Behaviour
alpha-carboxyl	2.19	loses H⁺ as pH rises
side-chain carboxyl	4.25	loses H⁺ as pH rises
alpha-amino	9.67	keeps H⁺ until pH passes it

Where it buffers

A weak acid buffers within about one pH unit either side of its pKa, which is the flat part of the curve. For glutamic acid that means:

Buffering range	Centred on
pH 1.19 to 3.19	pKa 2.19
pH 3.25 to 5.25	pKa 4.25
pH 8.67 to 10.67	pKa 9.67

Equivalence points

Halfway between two pKa values the previous group is fully titrated and the next has not started. Those are the steep parts.

Point	pH
Equivalence point 1	pH 3.22
Equivalence point 2	pH 6.96

What charge it carries, and when

Reading the curve left to right, each pKa you pass strips off one proton and drops the net charge by one. Between two pKa values one species dominates:

pH range	Net charge
below pH 2.19	+1
pH 2.19 to 4.25	0
pH 4.25 to 9.67	-1
above pH 9.67	-2

The charge is zero at the isoelectric point, which is why glutamic acid will not move in an electric field at pH 3.22. That is the basis of isoelectric focusing, and it is why the pI is the number worth remembering rather than the individual pKa values.

Net charge at physiological pH

At pH 7.4 no group is cleanly on or off. Each contributes a fraction, given by the Henderson-Hasselbalch relation, and the net charge is their sum:

the acid group (pKa 2.19) contributes -1.00
the acid group (pKa 4.25) contributes -1.00
the base group (pKa 9.67) contributes +0.99

Net charge on glutamic acid at pH 7.4 = -1.00

This is the calculation behind every peptide charge question. Do it for each residue in a sequence and add the results, and you have the charge on the whole peptide.

Working out the pI

Glutamic acid has three ionizable groups, so the pI is the average of the two pKa values that bracket the neutral form: (2.19 + 4.25) / 2 = 3.220, published as pI 3.22. The third group is already fully protonated or fully deprotonated there, so it does not enter the average.

Plot any amino acid's titration curve, or see all 20 amino acids with their pKa values. There is also a peptide charge and pI calculator for whole sequences.

Where the numbers come from

pKa values are the Lehninger set, the same ones used across this site. The curve is generated from those values with the Henderson-Hasselbalch relation for each group, and the pI is solved solved numerically as a check on the arithmetic above, which agrees to within 0.03 pH units.

Glutamic acid titration curve