Protein Isoelectric Point Calculator

The isoelectric point is the pH at which a polypeptide carries no net charge. Each ionizable group is treated independently with the Henderson-Hasselbalch relation, so a basic group contributes 1/(1 + 10^(pH - pKa)) of positive charge and an acidic group contributes the mirror term of negative charge. Seven side chains plus the two chain termini ionize in the accessible range. On the EMBOSS scale used here the values are aspartate 3.9, glutamate 4.1, cysteine 8.5, tyrosine 10.1, histidine 6.5, lysine 10.8, arginine 12.5, with the alpha-amino terminus at 8.6 and the alpha-carboxyl terminus at 3.6. Summing these curves gives a monotonically decreasing net-charge function, and because it crosses zero exactly once, bisection between pH 0 and 14 converges quickly on the pI. Proteome-wide the distribution of pI is bimodal, with peaks near pH 5 and pH 9 and a trough close to physiological pH 7.4, because proteins are least soluble at their own pI and cells appear to avoid that region. Most soluble cytosolic proteins fall between 4.5 and 7; histones and ribosomal proteins sit above 10, while pepsin is extreme at roughly 1. The practical uses are choosing ion-exchange chemistry (a protein binds an anion exchanger when the buffer pH exceeds its pI), predicting isoelectric precipitation, and placing spots on two-dimensional gels. Accuracy is limited: the pKa values come from free amino acids and ignore burial, salt bridges and local electrostatics, so measured pI often differs by 0.5 to 1 unit. Phosphorylation, sialylation and other post-translational modifications shift pI downward and are invisible to a sequence-only calculation, as are cysteines locked in disulfide bonds.

The charge-based separation of proteins began with Arne Tiselius at Uppsala, whose moving-boundary electrophoresis apparatus of 1937 first resolved serum proteins into albumin and the alpha, beta and gamma globulins and earned him the 1948 Nobel Prize in Chemistry. The concept of an isoelectric condition was older, but Tiselius made it measurable. Isoelectric focusing followed from theoretical work by Harry Svensson, later Rilbe, in the early 1960s, and became practical when Olof Vesterberg, also at Uppsala, synthesised carrier ampholytes in 1966 that could establish a stable pH gradient in a gel. Patrick O'Farrell combined focusing with SDS electrophoresis in 1975 to create two-dimensional gels, making pI a routinely measured protein property. Computation from sequence arrived with the sequence databases. Immobilized pH gradient strips introduced by Bjellqvist and colleagues in 1982 allowed precise experimental pI measurement, and in 1993 the same group published a set of pKa values fitted to observed migration in those gradients, which became the basis of the widely used Compute pI/Mw tool on the ExPASy server. A parallel scale ships with the iep program of the EMBOSS suite, released by Peter Rice, Alan Bleasby and colleagues around 2000 as an open-source successor to the GCG package, and it is that scale which this calculator applies. Later refinements attempt to model local electrostatic environment and structural context rather than treating each residue in isolation.