Isoelectric Point (pI) Calculator

Isoelectric Point (pI) Calculator

What is the Isoelectric Point (pI)?

The Isoelectric Point (pI) is the specific pH at which a particular molecule, such as an amino acid or protein, carries no net electrical charge. At a pH below its pI, the molecule will carry a net positive charge; at a pH above its pI, it will carry a net negative charge.

This property is crucial in biochemistry because it influences the solubility and behavior of proteins. Proteins are least soluble at their pI, a principle utilized in purification techniques like isoelectric focusing, where proteins migrate through a pH gradient until they reach the pH equal to their pI and precipitate out. The pI is determined by the pKa values of the ionizable groups within the molecule (the N-terminus, C-terminus, and acidic/basic side chains).

Frequently Asked Questions

How does pH affect a protein's charge?

Amino acids contain both acidic (carboxyl, -COOH) and basic (amino, -NH₂) groups, and some have ionizable side chains.
• At low pH (acidic conditions), there are many H⁺ ions. Both the amino group and carboxyl group are protonated (-NH₃⁺ and -COOH), resulting in a net positive charge (or less negative charge if acidic side chains are present).
• At high pH (basic conditions), H⁺ ions are scarce. Both groups are deprotonated (-NH₂ and -COO⁻), resulting in a net negative charge (or less positive charge if basic side chains are present).
• At the pI, the number of positive charges exactly balances the number of negative charges, resulting in a net charge of zero.

Why do different amino acids have different pKa values?

The pKa value indicates the acidity of a functional group (how easily it donates a proton). The exact pKa of a carboxyl, amino, or side chain group is influenced by its chemical environment. Factors like the electron-withdrawing or donating nature of nearby atoms, resonance stabilization, and local polarity within the amino acid structure cause slight variations in pKa values. For example, the carboxyl group's pKa is lower (more acidic) than a typical carboxylic acid because of the electron-withdrawing effect of the adjacent amino group.

How is the pI of a peptide calculated?

For a peptide, the pI depends on the pKa values of the N-terminal amino group, the C-terminal carboxyl group, and all ionizable side chains (Asp, Glu, His, Cys, Tyr, Lys, Arg). This calculator uses an iterative method: it calculates the peptide's net charge across a range of pH values. The pH where the net charge is closest to zero is determined to be the pI. This is visualized by plotting the net charge vs. pH, creating a titration curve.