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Biochemistry · Amino acids and proteins

Isoelectric point (pI) practice, with answers and a charge calculator

The isoelectric point (pI) is the pH where an amino acid has no net charge. The one big idea: find the neutral form, then average the two pKa values on either side of it. With that and a quick charge table, you can handle most pI and electrophoresis questions in college biochemistry.

Already know the basics? Jump to the 8 practice questions or the worked examples.

The rule: average the two pKa values around the neutral form

Amino acids have at least two ionizable groups: the α-carboxyl (COOH ⇌ COO⁻) and the α-amino group (NH₃⁺ ⇌ NH₂). Some side chains add a third. As pH rises, protons come off one at a time, from the lowest pKa to the highest. The isoelectric point (pI) is the pH where the average net charge is zero, which is where the neutral zwitterion form dominates.

pI = (pKa below the zwitterion + pKa above the zwitterion) / 2

  1. Neutral side chain: average the α-COOH and α-NH₃⁺ pKa values.
  2. Acidic side chain (Asp, Glu): average the two lowest pKa values. The zwitterion sits between losing the first and second carboxyl protons.
  3. Basic side chain (Lys, Arg, His): average the two highest pKa values. The zwitterion sits between losing the second and third protons.

A fast check: acidic amino acids have low pI values (around 3), basic ones have high pI values (around 7.6 to 10.8), and the rest sit near 5 to 6.3.

One pKa table, used everywhere on this page

pKa values vary slightly from one textbook to another, often by 0.1 or more, so your answer might differ a little from a classmate's. This page uses one source throughout: Table 26.1 in OpenStax Organic Chemistry (McMurry). Use your own course's table on exams.

Amino acid pKa values and pI, from OpenStax Organic Chemistry Table 26.1
Amino acidpKa α-COOHpKa α-NH₃⁺pKa side chainpI (listed)
Glycine (Gly)2.349.60—5.97
Alanine (Ala)2.349.69—6.01
Valine (Val)2.329.62—5.96
Leucine (Leu)2.369.60—5.98
Isoleucine (Ile)2.369.60—6.02
Methionine (Met)2.289.21—5.74
Proline (Pro)1.9910.60—6.30
Phenylalanine (Phe)1.839.13—5.48
Tryptophan (Trp)2.839.39—5.89
Serine (Ser)2.219.15—5.68
Threonine (Thr)2.099.10—5.60
Cysteine (Cys)1.9610.288.185.07
Tyrosine (Tyr)2.209.1110.075.66
Asparagine (Asn)2.028.80—5.41
Glutamine (Gln)2.179.13—5.65
Aspartic acid (Asp)1.889.603.652.77
Glutamic acid (Glu)2.199.674.253.22
Lysine (Lys)2.188.9510.539.74
Arginine (Arg)2.179.0412.4810.76
Histidine (His)1.829.176.007.59

Recomputing the listed pI values from the pKa columns with the averaging rule matches most of them to within 0.01. Two clearly do not: isoleucine (5.98 by the rule, 6.02 listed) and tryptophan (6.11 by the rule, 5.89 listed). The worked examples and questions here avoid those two, and the calculator reports the averaging-rule value.

Net charge at any pH, and electrophoresis

The charge table method is fast and needs no calculator. For each ionizable group, compare the pH with that group's pKa:

  1. pH well below the pKa: the group keeps its proton. COOH is 0, NH₃⁺ is +1.
  2. pH well above the pKa: the group loses its proton. COO⁻ is −1, NH₂ is 0.
  3. pH equal to the pKa: half and half, so the group contributes ±0.5.
  4. Add the charges. For a more exact value, use the Henderson-Hasselbalch fraction for each group, as the calculator below does.

Electrophoresis: at a pH below its pI, an amino acid or protein is net positive and moves toward the negative electrode (cathode). Above its pI, it is net negative and moves toward the positive electrode (anode). At its pI it does not move.

Worked examples

Example 1: pI of glycine

Glycine has pKa values of 2.34 (α-COOH) and 9.60 (α-NH₃⁺). Find its pI.

  1. The side chain is just H, so there are only two ionizable groups.
  2. Below pH 2.34 the form is +1. Between 2.34 and 9.60 it is the zwitterion, net 0. Above 9.60 it is −1.
  3. The two pKa values that flank the zwitterion are 2.34 and 9.60.
  4. pI = (2.34 + 9.60)/2 = 11.94/2 = 5.97.

Answer: pI = 5.97

Example 2: pI of aspartic acid, with a charge table

Aspartic acid has pKa values 1.88 (α-COOH), 3.65 (side-chain COOH), and 9.60 (α-NH₃⁺). Find its pI and its net charge at pH 7.

  1. Order the pKa values: 1.88, 3.65, 9.60. Fully protonated (pH below 1.88), the charge is +1.
  2. Lose the first proton (pKa 1.88): net 0. That is the zwitterion. Lose the second (pKa 3.65): net −1.
  3. The neutral form is flanked by 1.88 and 3.65, the two lowest values. pI = (1.88 + 3.65)/2 = 2.765, about 2.77.
  4. At pH 7: α-COOH is deprotonated (−1), side-chain COOH is deprotonated (−1), and α-NH₃⁺ is still protonated (+1). Net = −1.

Answer: pI ≈ 2.77; net charge at pH 7 is about −1.

Example 3: pI of lysine

Lysine has pKa values 2.18 (α-COOH), 8.95 (α-NH₃⁺), and 10.53 (side-chain NH₃⁺). Find its pI.

  1. Fully protonated, lysine has COOH (0) and two NH₃⁺ groups (+2). Net +2.
  2. Lose the proton at 2.18: net +1. Lose the proton at 8.95: net 0, the neutral form. Lose the proton at 10.53: net −1.
  3. The neutral form is flanked by 8.95 and 10.53, the two highest values.
  4. pI = (8.95 + 10.53)/2 = 19.48/2 = 9.74.

Answer: pI = 9.74

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Net charge calculator

Pick an amino acid and enter a pH. The calculator uses the OpenStax pKa values above and the Henderson-Hasselbalch fraction for each ionizable group, then adds up the charges.

Practice questions

Pick an answer, then check it. A wrong answer tells you what went wrong, so you can try again. Every question also has a full solution.

Question 1Glutamic acid has pKa values 2.19 (α-COOH), 4.25 (side chain), and 9.67 (α-NH₃⁺). What is its pI?

Show solution

Acidic amino acid, so average the two lowest pKa values: (2.19 + 4.25)/2 = 6.44/2 = 3.22.

Question 2Histidine has pKa values 1.82 (α-COOH), 6.00 (side chain), and 9.17 (α-NH₃⁺). What is its pI?

Show solution

Fully protonated histidine is +2. Losing the 1.82 proton gives +1, losing the 6.00 proton gives 0, and losing the 9.17 proton gives −1. The neutral form is flanked by 6.00 and 9.17: pI = 15.17/2 = 7.585 ≈ 7.59.

Question 3What is the approximate net charge of glycine (pKa 2.34 and 9.60) at pH 7.0?

Show solution

pH 7 is well above 2.34, so the carboxyl is COO⁻ (−1). It is well below 9.60, so the amine is NH₃⁺ (+1). Net 0. pH 7 is also fairly close to glycine's pI of 5.97, which fits.

Question 4In electrophoresis at pH 6.0, which way does lysine (pI 9.74) move?

Show solution

pH below pI means net positive. At pH 6.0 lysine has COO⁻ (−1) and two NH₃⁺ groups (+2), net about +1, so it moves toward the cathode.

Question 5What is the approximate net charge of aspartic acid (pKa 1.88, 3.65, 9.60) at pH 7.0?

Show solution

pH 7 is above 1.88 and 3.65, so both carboxyl groups are −1 each. It is below 9.60, so the amine is +1. Net −2 + 1 = −1.

Question 6Arginine has pKa values 2.17, 9.04, and 12.48 (side chain). What is its pI?

Show solution

(9.04 + 12.48)/2 = 21.52/2 = 10.76. Arginine has the highest pI of the 20 amino acids because its guanidinium group holds its proton so tightly.

Question 7Alanine has pKa values 2.34 and 9.69. What is its approximate net charge at pH 9.69?

Show solution

At pH = pKa, a group is 50% protonated. Carboxyl: −1. Amine: +0.5 on average. Net −0.5.

Question 8A mixture of lysine (pI 9.74), arginine (pI 10.76), histidine (pI 7.59), and glutamic acid (pI 3.22) is run in electrophoresis at pH 6.0. Which one moves toward the positive electrode?

Show solution

Compare pH with each pI. Only glutamic acid has pI below 6.0, so only it is negative (about −1) and moves toward the anode. The other three are positive and move toward the cathode.

Answer key

  1. B, 3.22: average of the two lowest pKa values.
  2. D, 7.59: average of the two highest pKa values.
  3. B, 0: zwitterion.
  4. C, toward the cathode: pH below pI, so positive.
  5. A, −1
  6. D, 10.76
  7. B, −0.5: half the amino groups deprotonated.
  8. C, glutamic acid: the only pI below the buffer pH.

Frequently asked questions

Is the isoelectric point on the MCAT?

Yes. Amino acid acid-base chemistry, including isoelectric points and how charge affects separation methods like electrophoresis, is part of the AAMC content outline. Expect to reason about charge at a given pH more than to memorize exact pI values.

Why do I get a slightly different pI than my textbook?

pKa values differ a little between sources, and rounding adds a few hundredths. Use the pKa table your course gives you. A difference of 0.01 to 0.1 usually just reflects the table.

Do cysteine and tyrosine need a special rule?

Their side chains are weakly acidic, but they ionize above the α-carboxyl pKa. Averaging the two lowest pKa values still gives the pI, and for tyrosine that is the same as averaging the two α groups. In the OpenStax table, cysteine's pI is 5.07 and tyrosine's is 5.66.

How does pI relate to proteins?

A protein also has a pI, set by all its ionizable side chains plus the two ends of the chain. Below its pI it is positive, above it negative. Isoelectric focusing separates proteins by moving each one to the pH where its net charge is zero.

Sources

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