, titration curve – – pKa (buffering region) – – pI
How to calculate pKa from a titration curve
Every pKa sits at a half-equivalence point, the flat middle of each buffering region. Read it in three steps:
It works because at half-equivalence exactly half the group is deprotonated, so [A-] = [HA]. Henderson-Hasselbalch becomes pH = pKa + log(1) = pKa. A curve with two flat regions has two pKa values, and the pI is the average of the two that flank the neutral form. For glycine, pKa 2.34 and 9.60 give pI = (2.34 + 9.60)/2 = 5.97.
How to read an amino acid titration curve
As you add base, you strip protons off the amino acid one group at a time. The flat, gently sloped stretches are buffering regions, each centered on a pKa. That is the half-equivalence point, where the group is half protonated and resists pH change best. The steep vertical jumps are equivalence points, where a proton has been fully removed. The isoelectric point (pI), where the molecule carries no net charge, sits at the midpoint of the two pKa values flanking the neutral zwitterion: for a simple amino acid like glycine that is just (pKa₁ + pKa₂)/2, and for one with an ionizable side chain you average the two pKas around the net-zero species.
pKa values are standard textbook values (Lehninger); sources vary by a few hundredths.
Want the by-hand method for finding pI? See how to calculate pI without a calculator.
Related tools: Peptide charge & pI calculator · Amino acid catabolism explorer · Acid-base titration curve · Amino acid chart (all 20) · all biochem tools.
Worked example 1: glycine, the simple 2-pKa case (default)
Glycine has no ionizable side chain, so it has just 2 pKa values: the carboxyl group (2.34) and the amino group (9.60).
This matches the calculator's default output exactly. This simple average is the version usually taught first. It works whenever there are exactly two ionizable groups.
Worked example 2: histidine, where you can't just average everything
Histidine has 3 pKa values: carboxyl (1.82), the imidazole side chain (6.00), and the amino group (9.17).
The carboxyl pKa of 1.82 is essentially irrelevant to the pI calculation here, by the time pH is anywhere near the isoelectric region, that group is already fully deprotonated and stays that way. Only the two pKa values that actually bracket the net-zero point (6.00 and 9.17) matter. Averaging all three pKas (which would give (1.82+6.00+9.17)/3 = 5.66) is a common mistake and gives the wrong answer, the tool correctly finds the true flanking pair by solving for where the net charge crosses zero, not by guessing which pKas to average.
Worked example 3: arginine, two basic groups
Arginine has 3 pKa values: carboxyl (2.17), the α-amino group (9.04), and the guanidino side chain (12.48), the most basic side chain of any standard amino acid.
Arginine's pI (10.76) is even higher than lysine's (9.74) because its guanidino side chain (pKa 12.48) is more basic than lysine's amine side chain (pKa 10.53). It holds onto its proton more strongly, so more base is needed to reach the neutral, net-zero point.
FAQ
What is a zwitterion?
A molecule carrying both a positive and negative charge at once, netting to zero. Amino acids at their pI are zwitterions: protonated amino group (positive), deprotonated carboxyl group (negative).
For 3 pKa values, why isn't pI the average of all three?
Only the two pKas directly flanking the neutral species matter, a third pKa far outside that range doesn't affect where the charge crosses zero, as shown in example 2 above. Averaging all three regardless is a common exam mistake.
Why do basic amino acids have pI above 7, acidic ones below?
An extra basic group needs more base (higher pH) to strip the last proton and reach net-zero, pushing pI up (lysine, arginine). An extra acidic group keeps the molecule net-negative until lower pH, pulling pI down (aspartate, glutamate).
Why does histidine's side-chain pKa matter so much?
It sits close to physiological pH (7.4, vs. histidine's ~6.0), so it's meaningfully both protonated and deprotonated near neutral pH, useful as a proton donor/acceptor in enzyme active sites and as a natural buffering component in proteins.
Practice problems
1. Lysine has three pKa values: 2.18 (carboxyl), 8.95 (α-amino), 10.53 (side-chain amine). What is its pI?
Show answer
2. Aspartate has pKa values 1.88 (carboxyl), 3.65 (side-chain carboxyl), 9.60 (α-amino). What is its pI?
Show answer
Sources and how to cite this page
The pKa values used to draw these curves are the standard set tabulated in Lehninger Principles of Biochemistry (Nelson & Cox), the same set used in most biochemistry courses and on the MCAT. Glycine, for example, is given here as pKa1 2.34 (α-carboxyl), pKa2 9.60 (α-amino), and pI 5.97, where the pI is the average of the two pKa values that flank the zwitterion. Each curve is computed from the Henderson-Hasselbalch equation rather than drawn by hand, so the half-equivalence points fall exactly on the pKa values.
Why published pKa values differ slightly. pKa is measured, not derived, so a tabulated value depends on temperature and ionic strength. Values here are for dilute aqueous solution near 25 °C. Differences of roughly 0.1 to 0.3 pKa units between references are normal and do not mean one is wrong. If you are citing a number for coursework, cite the source you were taught from and stay consistent with it.