Charge vs pH

The titration curve of the protein — net charge across the pH range, with the isoelectric point where it crosses zero and the charge at the pH you actually work at.

What it doesCopy Anchor Link

The isoelectric point is the single number everyone quotes, and it is one point on this curve. It tells you where the protein has no net charge, and nothing about how much charge it carries anywhere else. Two proteins with the same pI can differ by ten units of charge at pH 7, because pI depends on where the ionisable groups balance and net charge depends on how many there are.

This is the curve. Net charge is summed over every ionisable group — the side chains and the two termini — at each pH, and drawn across the range. The pI is where it crosses zero, marked. The charge at a pH you choose is read off directly, interpolated between the computed points rather than rounded to the nearest one.

The slope matters as much as the crossing. A curve that is steep through your working pH is a protein whose charge changes sharply with a small buffer error; a flat one is forgiving.

When you'd use thisCopy Anchor Link

• Before ion-exchange chromatography. Which resin, and at what buffer pH, is decided by the sign and the size of the charge at that pH — not by the pI.
• When a protein precipitates. Aggregation near the pI is the classic case, and the curve says how near you are and how fast you leave.
• To choose a buffer. Working at a pH where the curve is steep means small pH drifts become charge changes.
• When a mutation changes the surface. Removing or adding a charged residue shifts the whole curve, and the shift at your working pH is the quantity of interest.

What this computesCopy Anchor Link

• Net charge as a function of pH, from the Henderson–Hasselbalch contribution of every ionisable group in the sequence plus the free amino and carboxyl termini.
• The isoelectric point, the pH at which that sum is zero.
• The charge at a chosen pH, interpolated across the computed grid so an arbitrary pH gives a real number rather than the nearest grid point's.
• The counts behind it — how many acidic and how many basic residues the charge is being summed over, which is what makes an unexpected curve traceable to the sequence.

What it does not doCopy Anchor Link

It is a sequence calculation using standard side-chain pKa values, so it treats every residue of a kind as identical. In a folded protein they are not: a buried aspartate, one in a salt bridge, and one on the surface have measurably different pKa values, which is why a measured pI can differ from a calculated one by a fraction of a unit or more. It does not model the fold, ionic strength, bound ligands, or post-translational modifications such as phosphorylation, each of which moves the curve.