# How to calculate isoelectric point

An understanding of how to calculate isoelectric point is essential when you are working with proteins. The isoelectric point is the pH at which the net electrical charge on a molecule is zero. The pI value helps you determine the solubility of a molecule at a given pH.  The isoelectric point of proteins plays a significant role in the separation and purification methods used to isolate proteins from a solution. For instance, in isoelectric focusing, a pH gradient is used to separate proteins based on their isoelectric points.

This article will explain what is the isoelectric point, the formula used to calculate isoelectric point, and examples to demonstrate how the formula may be applied.

## What is an isoelectric point?

The isoelectric point is the pH of a solution at which an amino acid or peptide is neutral and carries zero net electric charge. An aminoacid carrying no net charge is called a zwitterion. The zwitterion is the electrically neutral form of amino acid, that carries a negatively charged carboxylate group (CO2 –) and a positively charged amino group (NH3+ ). The two opposite charges cancel out giving the zwitterion a net charge of zero.  In an aqueous solution having a pH equal to the isoelectric point, most amino acids are present in the solution as zwitterions. At this pH, the aminoacids show minimum solubility.

The zwitterion structure of Alanine. Amino acids interact intramolecularly to form zwitterions. At a neutral pH, an acid-base reaction occurs between the carboxyl group and the amino group of the amino acid. The carboxyl group loses its hydrogen that is taken up by the amino group to form ammonium. The positively charged ammonium balances the negative charge of the carboxylate.

## Isoelectric point Formula

The isoelectric point formula for an amino acid having a neutral side chain will be as follows:

pI=pKa1  +pKa22

pKa1 – pKa of the carboxylate group of amino acid

pKa2 –pKa of the amino group of the amino acid

For amino acids having a neutral side chain, the pI value is calculated by taking a mean of the pKa of the carboxylate and the pKa of the aminonium group. For amino acids having a charged or ionizable side chain, the pI value is calculated by taking a mean of the pKas’ of similarly ionizable groups. This will be explained further in the examples section.

## Isoelectric Point Definition

The isoelectric point, pI, is defined as the pH of an aqueous solution of an amino acid at which the amino acids in the solution carry zero net charge. This means that at the isoelectric point, the positively charged groups in the solution are balanced by the negatively charged groups.  The amino acids exist as zwitterions in an aqueous solution that has a neutral pH.

A diagram representing the effect of pH on the net charge of a neutral amino acid.

## Examples

• Calculate the isoelectric point of Alanine

Solution:

Since Alanine has a neutral side chain (-CH3), the isoelectric point will be calculated using the pKa values of the carboxylic acid group and the ammonium group:

pI=2.34  +9.692=6.02

• Calculate the isoelectric point of Aspartic acid

Solution:

Aspartic acid is an amino acid having an acidic side chain CH2COOH. To calculate the isoelectric point for this amino acid, the pKa of the similarly ionizable groups will be considered which in this case are the carboxylic acid group (pKa = 1.88) and the acidic side chain (pKa = 3.65).

pI=1.88 +3.652=2.77

• Calculate the isoelectric point of Lysine

Solution:

Lysine has a basic group in its side chain. To calculate the isoelectric point of Lysine, the pKa of the similarly ionizable groups will be considered which are the ammonium group (pKa = 8.95) and the basic side chain (pKa = 10.53).

pI=8.95+10.532=9.74

### Isoelectric points of acidic and basic amino acids

The isoelectric point of amino acids differs based on the following factors:

• Sidechain groups present in an amino acid determine the isoelectric point of that amino acid. The isoelectric point of each amino acid is different due to the presence of a unique side chain group, usually represented with the letter R.
• The isoelectric point of neutral amino acids is near pH 7.
• Amino acids having acidic side chains have isoelectric points less than 7. This is because at pH 7 the acidic amino acid’s side chain carboxylic acid group exists as a carboxylate ion, so it has a net negative charge. To balance the negative charge in the side chain, carboxylate ion has to be protonated. Thus, the solution has to be acidic to protonate the carboxylate group in the side chain.
• Basic amino acids tend to have an isoelectric  point greater than 7. This is because the basic group in the side chain is protonated at pH 7. This means that two ammonium ions and only one carboxylate ion are present in the amino acid at ph7 giving it a net charge of +1.  To neutralize the +1 charge on the amino acid, the ammonium group in the side change needs to be deprotonated. At a pH higher than 7, more OH- ions will trigger the deprotonation of the amino group in the side chain.

A table showing the pKa values and pI values of 20 naturally occurring amino acids.

pKa1= α-carboxyl group, pKa2 = α-ammonium ion, and pKa3 = side chain group.

## Isoelectric points and electrophoresis

Electrophoresis in general refers to the separation of molecules based on their net charge under the influence of an electric field. At a given pH, an amino acid will exist in either positively or negatively charged forms. When a mixture of different amino acids is placed on a fluid or paper and a constant electric field is applied, the amino acids having a net negative charge will move towards the anode (positive electrode) while the positively charged amino acids will move towards the cathode (negative electrode). Thus electrophoresis can separates amino acids based on their net charge at a particular pH.

Isoelectric focusing in particular is a technique involving electrophoresis that separates proteins based on their isoelectric points. An electric field is applied to the mixture of proteins within a pH gradient. The proteins travel through the pH gradient when a voltage is applied. Once a protein has reached a pH value that is equal to its isoelectric point (pI) , the net electrical charge on that protein becomes neutral, and the amino acid stops moving in the gel. Thus, each protein in the sample is separated based on the isoelectric point.

A diagram depicting isoelectric focusing for the separation of proteins.

## Further Problem Questions involving Isoelectric point

Problem 1.

A mixture containing glutamic acid (pI = 3.2), arginine (pI = 10.8), and valine (pI = 6.0), is subjected to electrophoresis.  If electrophoresis is carried out at a pH of 7.2:

a)which amino acid will move towards the cathode (-vely charged)?

(b) Which amino acids will move towards the anode(positively charged)?

(c) Which amino acid will move farthest toward the anode?

Solution:

a) Arginine, pH applied is lesser than the isoelectric point of arginine; arginine will be protonated and will carry a net positive charge. Thus, Arginine will migrate towards the cathode.

b) Valine and Glutamic acid, since both will be deprotonated and will have a net negative charge.

c) Glutamic acid, because it carries a greater negative charge due to a greater difference between its pI and the applied pH.

Problem 2.

Estimate the isoelectric points of each of the following tripeptides.

1. Ala-Val-Gly

Isoelectric point will be near pH 7, because the amino acid residues present in this tripeptide are all neutral amino acids.

1. Ser-Val-Asp

The pI for this tripeptide will be less than 7, due to the presence of an acidic amino acid (Asp) in this tripeptide.

1. Lys-Ala-Val

The pI for this tripeptide is greater than 7, because a basic amino acid (Lys) is present in the tripeptide.

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