Isoelectric Point (pI) Calculator for Amino Acids

The isoelectric point is the pH at which the summed positive and negative charges on an amphoteric molecule cancel. For an amino acid where only the alpha-carboxyl and alpha-amino groups ionise, pI is the arithmetic mean of the two pKa values, giving 5.97 for glycine from 2.34 and 9.60. When the side chain also ionises, the correct pair to average is the two pKa values that bracket the neutral species: the two lowest for acidic residues, so aspartate with 2.09, 3.86, and 9.82 has a pI near 2.98, and the two highest for basic residues, so lysine with 2.18, 8.95, and 10.53 has a pI of 9.74. Below its pI a molecule is net positive and migrates toward the cathode, and above it the molecule is net negative and moves toward the anode. The practical consequence is a solubility minimum at the pI, where the loss of electrostatic repulsion lets molecules associate. This is the basis of isoelectric precipitation and of the rule that protein formulations are buffered at least one pH unit away from pI. Whole proteins cluster mostly between pI 5 and 9, with histones above 10 and pepsin near 1, reflecting the acidic stomach in which it works. Simple averaging of tabulated pKa values is only an approximation for proteins: neighbouring charges, hydrogen bonding, burial of a residue in the hydrophobic interior, and post-translational modifications such as phosphorylation can each shift an individual pKa by more than a full unit, so calculated and observed pI values commonly differ by 0.3 to 1.0 pH units.

The idea that proteins carry a pH-dependent charge with a neutral crossing point emerged from colloid chemistry at the turn of the twentieth century. William Bate Hardy, working in Cambridge from 1899, showed that globulins migrate in an electric field in a direction that reverses with acidity and that they coagulate most readily at the reversal point. Leonor Michaelis, better known for enzyme kinetics, systematised the concept and its terminology in a series of papers on the isoelectric point during the 1910s, once Soren Sorensen had introduced the pH scale in 1909 to give the axis a proper definition. Measurement improved through electrophoresis. Arne Tiselius built the moving boundary apparatus at Uppsala in 1937 that separated serum proteins into albumin and the alpha, beta, and gamma globulin fractions, work recognised with the Nobel Prize in Chemistry in 1948. Harry Svensson, later known as Rilbe, worked out the theory of steady-state isoelectric focusing in a pH gradient in 1961 and 1962, and Olof Vesterberg synthesised the carrier ampholytes in 1969 that made it practical. Patrick O'Farrell combined focusing with molecular weight separation in 1975 to create two-dimensional gel electrophoresis, and the immobilised pH gradients introduced by Bjellqvist and co-workers in 1982 gave the reproducibility that made pI a routine protein identifier.