Acid-Base Chemistry of Amino Acids
Amino acids are amphoteric, shifting between charged and neutral zwitterion forms depending on how pH compares to each ionizable group's pKa.
Amino acids are amphoteric — they can act as either an acid or a base, gaining or losing protons depending on the pH of their environment. Every amino acid has at least two ionizable groups, a carboxyl group and an amino group, and how those groups are charged at any given moment depends entirely on where the surrounding pH sits relative to each group's pKa.
Key Takeaways
Amino acids are amphoteric, existing as zwitterions (net neutral) near physiological pH.
Every amino acid has at least two pKa values (~2 for the carboxyl group, ~9-10 for the amino group); ionizable side chains add a third.
Below a group's pKa, it's mostly protonated; above it, mostly deprotonated.
The isoelectric point (pI) is the pH of zero net charge; it's calculated by averaging the two pKa values that flank the zwitterion.
For amino acids without a side-chain pKa (like glycine), that's simply the average of the two backbone pKa values. For amino acids with a side-chain pKa (like glutamic acid or lysine), it's the average of whichever two pKa values surround the neutral zwitterionic form.
Amino Acids Are Amphoteric
At the most basic structural level, every amino acid has a carboxyl group and an amino group capable of gaining or losing a proton. Written with no protons gained or lost, these would appear as –COOH and –NH₂. But that exact form rarely exists in water — especially near physiological pH.
The Zwitterion: Why Amino Acids Carry No Net Charge at Neutral pH
In water, the carboxyl group tends to lose a proton, becoming negatively charged (–COO⁻), while the amino group tends to gain a proton, becoming positively charged (–NH₃⁺). The result is a zwitterion — a molecule carrying both a positive and a negative charge on different atoms, but with no net charge overall. This zwitterionic form is the dominant state for most amino acids at physiological pH (about 7.4).
pKa and Protonation State
Two rules govern how ionizable groups behave:
Ionizable groups gain protons under acidic conditions and lose them under basic conditions. At low pH, groups tend to be protonated; at high pH, they tend to be deprotonated.
The pKa of a group is the pH at which half of that group's molecules are deprotonated. Below the pKa, the group is mostly protonated. Above the pKa, it's mostly deprotonated.
Every amino acid has at least two pKa values:
pKa1, usually around 2, for the alpha-carboxyl group.
pKa2, usually around 9 to 10, for the alpha-amino group.
If the amino acid also has an ionizable side chain (like aspartate, glutamate, lysine, arginine, histidine, cysteine, or tyrosine), it has a third pKa specific to that side chain.
Amino Acid Charge Across the pH Scale
pH Range | Carboxyl Group | Amino Group | Net Charge |
|---|---|---|---|
Low (acidic) | –COOH (protonated) | –NH₃⁺ (protonated) | Positive |
Near physiological pH (~7.4) | –COO⁻ (deprotonated) | –NH₃⁺ (protonated) | Neutral (zwitterion) |
High (basic) | –COO⁻ (deprotonated) | –NH₂ (deprotonated) | Negative |
The Isoelectric Point (pI) and Titration Curves
The isoelectric point (pI) is the pH at which an amino acid carries no net charge — where it exists entirely as a zwitterion. Because amino acids have both acidic and basic ionizable groups, they make excellent subjects for titration experiments, which reveal exactly how their charge shifts as pH changes.
Glycine's Titration Curve (Worked Example)
Titrating a fully protonated glycine solution with base plays out in four stages:
Fully protonated start. At low pH, glycine's carboxyl group is –COOH and its amino group is –NH₃⁺, giving it a net positive charge.
First buffering region. As base is added, the carboxyl group (lower pKa, more acidic) loses its proton first. At pKa1 = 2.34, the protonated and deprotonated forms of the carboxyl group are in equilibrium, and the curve flattens because glycine buffers around this pKa.
The isoelectric point. Continued addition of base brings the solution to pH 5.97, glycine's pI, where it exists entirely as the neutral zwitterion. Because the molecule carries no net charge here, it no longer buffers, and the curve rises steeply.
Second buffering region. Around pKa2 = 9.60, the amino group loses its proton (–NH₃⁺ → –NH₂), and the zwitterion and fully deprotonated forms coexist in equilibrium.
Calculating pI for Amino Acids Without an Ionizable Side Chain
For an amino acid like glycine, with only two ionizable groups, the pI is the average of the two pKa values:
pI = (pKa1 + pKa2) / 2 = (2.34 + 9.60) / 2 = 5.97
Calculating pI for Amino Acids With an Ionizable Side Chain
Amino acids with a third, ionizable side-chain group have three pKa values and a slightly different rule: the pI is the average of the two pKa values that flank the neutral zwitterionic form — not simply the two lowest or two highest values.
Glutamic acid has two carboxyl groups (backbone and side chain) plus one amino group. Its zwitterion forms after the first carboxyl group deprotonates but before the side-chain carboxyl group does, so the pI averages those two carboxyl pKa values: pKa1 = 2.19 and pKa(R) = 4.25, giving pI = 3.22.
Lysine has two amino groups (backbone and side chain) plus one carboxyl group. Its zwitterion exists after the carboxyl group deprotonates but before either amino group does, so the pI averages the two amino pKa values — giving pI ≈ 9.74.
Amino Acid | pKa1 (α-COOH) | pKa(R) (side chain) | pKa2 (α-NH₃⁺) | pI |
|---|---|---|---|---|
Glycine | 2.34 | — | 9.60 | 5.97 |
Glutamic acid | 2.19 | 4.25 | 9.67 | 3.22 |
Lysine | 2.18 | 10.53 | 8.95 | 9.74 |
MCAT Callout — pI Shortcut Rule: Whether an amino acid has two or three pKa values, its pI is always the average of the two pKa values immediately surrounding its zwitterionic form on the titration curve.
Common MCAT Mistakes
Averaging the wrong two pKa values. For amino acids with an ionizable side chain, students default to averaging the highest and lowest pKa (or the two backbone pKa values). The correct rule is always to average the two values flanking the zwitterion — for glutamic acid that means the two carboxyl pKa values, not one carboxyl and the amino group.
Forgetting a side chain adds a third pKa. Amino acids like lysine, arginine, histidine, cysteine, tyrosine, aspartate, and glutamate each have three ionizable groups, not two — missing the side-chain pKa leads to using the two-pKa (glycine-style) formula incorrectly.
Assuming the zwitterion means "uncharged everywhere." A zwitterion has zero net charge, but it still carries a full positive charge (–NH₃⁺) and a full negative charge (–COO⁻) simultaneously — not a molecule with no charged atoms at all.
Confusing pKa with pH. The pKa is a fixed property of a specific ionizable group; pH is the property of the surrounding solution. Whether a group is protonated or deprotonated depends on comparing the solution's pH to that group's pKa, not on the pKa value alone.
MCAT-Style Concept Check
Question: Aspartic acid has three pKa values: pKa1 (α-carboxyl) = 1.88, pKa(R) (side-chain carboxyl) = 3.65, and pKa2 (α-amino) = 9.60. What is aspartic acid's isoelectric point (pI)?
A) 1.88, because the pI is always equal to the lowest pKa value
B) 2.77, because the pI is the average of the two carboxyl pKa values, which flank its zwitterionic form
C) 5.74, because the pI is the average of the highest and lowest pKa values
D) 6.63, because the pI is the average of the side-chain pKa and the amino group pKa
Answer: B
Explanation: Aspartic acid's zwitterion forms after both carboxyl groups (α and side-chain) have deprotonated but before the amino group does — at that point the molecule carries one negative charge (from whichever carboxyl group deprotonates last) balanced by the protonated amino group. The pI is the average of the two pKa values flanking that zwitterionic form: (1.88 + 3.65) / 2 = 2.77. Options A, C, and D apply the wrong pair of pKa values instead of identifying which two actually flank the neutral zwitterion.
FAQ
What does it mean for an amino acid to be amphoteric?
It means the amino acid can act as either an acid or a base — its carboxyl group can donate a proton and its amino group can accept one, depending on the surrounding pH.
What is a zwitterion?
A zwitterion is a molecule that carries both a positive charge and a negative charge on different atoms at the same time, resulting in zero net charge overall. Most amino acids exist as zwitterions at physiological pH.
How do you calculate an amino acid's isoelectric point (pI)?
Average the two pKa values that flank the amino acid's neutral zwitterionic form. For amino acids without an ionizable side chain (like glycine), that's simply the two backbone pKa values. For amino acids with an ionizable side chain (like glutamic acid or lysine), it's whichever two of the three pKa values surround the zwitterion.
Why do glutamic acid and lysine use different pairs of pKa values to calculate pI?
Because their zwitterions form at different points on the titration curve. Glutamic acid's zwitterion sits between its two carboxyl pKa values, so those two are averaged. Lysine's zwitterion sits between its two amino pKa values, so those two are averaged instead.