Isoelectric Point Calculators
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Calculating pI for Amino Acids
For a simple diprotic amino acid (pKa₁ = carboxyl, pKa₂ = amino): pI = (pKa₁ + pKa₂) / 2
For glycine: pKa₁ = 2.35; pKa₂ = 9.60 → pI = (2.35 + 9.60) / 2 = 5.97.
For acidic amino acids (Asp, Glu) with a side chain carboxyl: pI = (pKa₁ + pKa_side_chain) / 2 (the two acidic groups). For basic amino acids (Lys, Arg, His): pI = (pKa₂ + pKa_side_chain) / 2.
Protein pI
Protein pI depends on its full amino acid composition — the number and pKa values of all ionizable residues (Asp, Glu, His, Cys, Tyr, Lys, Arg, N-terminus, C-terminus). The Henderson-Hasselbalch equation is applied iteratively to find the pH where sum of positive charges = sum of negative charges. Most intracellular proteins have pI 5–7; secreted proteins are often more basic (pI 7–11).
Isoelectric Focusing (IEF)
IEF separates proteins by pI in a pH gradient gel. Proteins migrate under electric field until they reach the pH matching their pI, where they stop (net charge = 0). Resolution can distinguish proteins differing by 0.01 pH unit. IEF is the first dimension in 2D gel electrophoresis (2D-PAGE), followed by SDS-PAGE for molecular weight separation.
Protein Solubility at pI
Proteins are least soluble at their pI because with zero net charge, electrostatic repulsion between molecules is minimized, promoting aggregation and precipitation. This is exploited in isoelectric precipitation to purify proteins from complex mixtures.
Glossary
Frequently Asked Questions
The isoelectric point (pI) is the pH at which a protein has zero net charge — positive charges on basic residues (Lys, Arg, His) exactly balance negative charges on acidic residues (Asp, Glu) and the termini. Below the pI, the protein is positively charged; above the pI, negatively charged. Most intracellular proteins have pI in the 5–7 range. pI can be estimated from amino acid sequence using ExPASy ProtParam tool (web.expasy.org/protparam).
For a simple diprotic amino acid: pI = (pKa₁ + pKa₂) / 2, where pKa₁ is the carboxyl group and pKa₂ is the amino group. Glycine: pI = (2.35 + 9.60)/2 = 5.97. For acidic amino acids like aspartate (pKa₁ = 2.1, pKa_side = 3.9, pKa₂ = 9.8): pI = (pKa₁ + pKa_side)/2 = (2.1 + 3.9)/2 = 3.0. For lysine (pKa₁ = 2.2, pKa₂ = 9.2, pKa_side = 10.5): pI = (pKa₂ + pKa_side)/2 = (9.2 + 10.5)/2 = 9.85.
IEF separates proteins by their pI in a pH gradient — typically a polyacrylamide gel containing ampholytes that establish a stable pH gradient under electric current. Proteins migrate through the gradient until they reach the zone where pH = pI, at which point they carry no charge and stop moving. Proteins are separated by pI, not size. IEF can resolve proteins differing by just 0.01 pH unit. It is used as the first dimension of 2D-PAGE, which combines IEF (by pI) with SDS-PAGE (by molecular weight) to create a 2D protein map.
At the pI, a protein carries zero net charge. Without electrostatic repulsion to keep molecules apart, proteins can come into close contact and interact through hydrophobic and van der Waals forces, promoting aggregation and precipitation. Moving the pH away from the pI gives the protein a net charge that causes electrostatic repulsion between molecules, increasing solubility. This property is used in isoelectric precipitation — adjusting pH to a protein's pI to selectively precipitate it from a mixture, as done historically in casein precipitation from milk.