Isoelectric Point Calculator
Find the pH at which a molecule or protein carries no net charge, from the pKa values of its ionisable groups.
What it does
Proteins are sorted in a lab by charge, and the number that decides the sorting is the pH at which the molecule stops carrying a net charge. That pH is the isoelectric point, and it is the one property of a protein that you can predict from a short list of numbers.
The organic chemistry text behind this page explains the picture in terms of the Henderson-Hasselbalch equation. In acid solution an amino acid is protonated and carries a positive charge, in basic solution it is deprotonated and carries a negative charge, and in between there is a pH at which the two forms balance and the molecule sits as the neutral dipolar zwitterion. That pH is the isoelectric point, and the same text gives it a value of 6.01 for alanine.
The rule for finding it is the one the textbook gives: the pI of an amino acid is the average of the two acid dissociation constants that involve the neutral zwitterion. For the 13 amino acids with a neutral side chain that is the average of pKa1 and pKa2. For the four with an acidic side chain it is the average of the two lowest pKa values, and for the three with a basic side chain it is the average of the two highest.
This page takes the general form of that rule, so it works for proteins as well as single amino acids. Give it each ionisable group with its pKa value and the charge it carries when protonated, and it finds the pH at which the net charge passes through zero, reports the two constants that bracket it, and gives you the net charge at pH 7 so you can see which way the molecule would move in a field.
The limits are worth stating. The result depends on the pKa values you feed in, and those shift with temperature, with ionic strength and with the environment of the group inside a folded protein. The rule also assumes the groups behave independently, which is an approximation for anything but the simplest molecules.
How to use
- List every group that can gain or lose a proton, as pKa:charge, separated by commas.
- Use 0 for the charge of a protonated carboxyl group, +1 for a protonated ammonium or guanidinium group, and -1 for a group that is already negative when protonated.
- For a single amino acid there are two or three groups. For a protein, use the pKa values of the groups that ionise in the pH range you care about.
- Read the isoelectric point and the two pKa values that bracket it.
- Read the net charge at pH 7 to see which way the molecule would drift in an electric field.
- Use the value to set the buffer for electrophoresis or for any separation step, and remember that the pKa values shift with the conditions.
Formula
- pI
- Isoelectric point, the pH at which the net charge of the molecule is zero.
- pKa
- Acid dissociation constant of one group, written as the pH at which that group is half protonated.
- q
- Charge a group carries when it is protonated, so 0, +1 or -1.
- f
- Fraction of a group that is still protonated at the pH in question, from the Henderson-Hasselbalch equation.
Each group that loses a proton drops the charge of the molecule by one, so the net charge starts at the sum of the protonated charges and falls step by step as the pH rises past each pKa value. The isoelectric point is the middle of the region where the net charge is zero, which is why it is the average of the two pKa values on either side of that region.
Worked example
Example: alanine with a pKa of 2.34 for the carboxyl and 9.69 for the ammonium
Defaults: two groups, 2.34:0 and 9.69:+1.
- The two groups are the carboxyl group with a pKa of 2.34 and a protonated charge of 0, and the ammonium group with a pKa of 9.69 and a protonated charge of +1.
- The molecule starts with a net charge of +1 in strong acid, because only the ammonium group is protonated.
- Above pKa 2.34 the carboxyl loses its proton, so the net charge falls to 0 and the molecule is the neutral zwitterion.
- Above pKa 9.69 the ammonium loses its proton too, so the net charge falls to -1.
- The net charge is zero between the two constants, so the isoelectric point is (2.34 + 9.69) / 2 = 6.02.
- At pH 7 the carboxyl is fully deprotonated and the ammonium is 99.8 percent protonated, so the net charge works out at -0.002.
- The tool shows 6.02, the bracketing values 2.34 and 9.69, a net charge of 0.000 at that pH, a net charge of -0.002 at pH 7, and the two groups it used.
Reference table
| Amino acid | pKa values | Isoelectric point | Net charge at pH 7 |
|---|---|---|---|
| Alanine | 2.34 and 9.69 | 6.02 | -0.002 |
| Aspartic acid | 2.1, 3.9 and 9.8 | 3.00 | -1.001 |
| Histidine | 1.8, 6.0 and 9.2 | 7.60 | 0.085 |
| Arginine | 2.1, 9.0 and 12.5 | 10.75 | 0.990 |
| Lysine | 2.2, 9.0 and 10.5 | 9.75 | 0.990 |
| Cysteine | 1.7, 8.3 and 10.4 | 5.00 | -0.048 |
Limits
- The pKa values depend on temperature, on the ionic strength of the buffer and on the surroundings of the group, and they shift when a protein folds.
- The rule treats each group as independent, which is a fair assumption for single amino acids and only an approximation for a folded protein.
- It gives one number for the whole molecule. A protein with several domains can have more than one charge state and no single clean pI.
- It says nothing about the solubility or the stability of the molecule, only about its charge.
Common mistakes
- Averaging the wrong two pKa values. For an acidic side chain the two lowest values are the ones that matter, and for a basic side chain the two highest.
- Forgetting to include the charge of each group, so the running total of the net charge starts in the wrong place.
- Leaving out a side chain group, which moves the pI by more than a unit in most cases.
- Using pKa values measured in a different buffer, where the ionic strength can shift them by a few tenths of a unit.
- Expecting the pI to be the pH at which the molecule is least soluble in every solvent. It is the point of zero charge, and solubility follows from that only in simple cases.
Try it yourself
Quick checks. Answers sit tucked under each question.
What is the isoelectric point of aspartic acid?
3.00. Its groups are the carboxyl at pKa 2.1, the side chain carboxyl at 3.9 and the ammonium at 9.8, and the net charge reaches zero between the two carboxyl groups, so the pI is (2.1 + 3.9) / 2 = 3.00. At pH 7 it carries a net charge of -1.001.
And histidine, which has a basic side chain?
7.60, from (6.0 + 9.2) / 2. The imidazole group of the side chain and the ammonium bracket the zero charge region, and because the pI is above 7 the molecule is still slightly positive at pH 7, with a net charge of 0.085.
What happens to a protein that is moved from below its pI to above it?
Its net charge swings from positive to negative, so it reverses direction in an electric field. That is the whole basis of electrophoresis: the text behind this page describes proteins above their pI as deprotonated and migrating towards the positive electrode, and proteins below their pI as protonated and migrating the other way.
Questions
What is an isoelectric point?
The pH at which a molecule or protein carries no net charge. The organic chemistry text behind this page calls it the pH at which the positively charged groups are exactly balanced by the negatively charged ones.
How do I calculate the pI of an amino acid?
Take the two pKa values that involve the neutral zwitterion and average them. For a simple amino acid with a neutral side chain that is pKa1 and pKa2, and the alanine example gives (2.34 + 9.69) / 2 = 6.02.
Why do the acidic and basic amino acids differ?
The side chain changes which pair of constants brackets the neutral form. An acidic side chain deprotonates early, so its pI comes from the two lowest pKa values, and a basic side chain stays protonated, so its pI comes from the two highest.
How do I find the pI of a protein?
List the pKa values of the groups that ionise in the range of interest, with the charge each one carries when protonated, and let the tool find the pH where the net charge passes through zero. The same source notes that a protein with more basic amino acids has a higher pI, with lysozyme at 11.0 and pepsin at about 1.0.
What is the charge of a molecule at pH 7 relative to its pI?
Above its pI the molecule is on average negative, and below its pI it is on average positive. That is why the page also reports the net charge at pH 7.
Why does the pI matter in a lab?
Solubility in water is usually lowest at the isoelectric point, because the molecule has no net charge to hold it in solution, and the charge is what drives the movement in electrophoresis. Both effects are described in the source behind this page.
Sources
- Chemistry LibreTexts, OpenStax organic chemistry 26.2: the Henderson-Hasselbalch equation and isoelectric points
- Chemistry LibreTexts, organic chemistry III 26.3: pKa values of polyfunctional amino acids and the pI rule