LSM2106 Chap.3 Amino Acids and Their Ionisation
Amino Acids and Their Ionisation
Three pK values per residue, and only one of them varies usefully
The third lecture introduces amino acids, and the course supplies its own table of pK values for all twenty residues found in proteins. Every residue carries a backbone carboxyl group and a backbone amino group; seven of them carry a third ionisable group on the side chain.
The table assigns the first pK to the carboxyl and the second to the amino group, and gives a third value only where the side chain has one.
The backbone values hardly move across the twenty residues, roughly between 1.8 and 2.6 for the carboxyl and between 8.8 and 10.8 for the amino group.
At any pH near neutrality the backbone of a free amino acid is therefore a zwitterion, with a negative carboxylate and a positively charged amino group. All the chemistry that distinguishes one residue from another sits in the side chain.
Aspartate and glutamate are negative well below neutrality, lysine and arginine are positive well above it, tyrosine and cysteine change state in the alkaline range, and histidine alone switches close to physiological pH.
That last fact explains both why histidine turns up in so many active sites and why the first practical chooses it for titration.
Reading a titration, and computing an isoelectric point
Titrating a residue with more than one ionisable group produces a curve with one step per group. Each plateau is a buffering region, and the pH at the midpoint of a step is the pK of the group being titrated there.
The practical asks for the value to be read in two ways: as the pH after half an equivalent of base has been added, and as the point of inflexion, which is the centre of symmetry of the step.
The second route is more robust when the concentration of the titrant is uncertain, because it does not depend on knowing how much base was added.
Plotting pH against moles of base per mole of acid rather than against volume removes both the volume and the concentrations from the picture, so curves from different groups become comparable and the midpoints fall at predictable positions.
The isoelectric point is the pH at which net charge is zero, and it is the average of the two pK values that bracket the neutral species, not an average of all three.
Writing out the species in order of increasing pH, marking the one whose charges cancel and averaging the two values on either side of it is the procedure that prevents the standard error here.
The direction rule underpins everything: a group is protonated below its own pK and deprotonated above it, and whether that protonation produces a charge depends on whether the group is an acid or a base.
What this chapter covers
- 01
The backbone carboxyl and amino groups, and the narrow range their pK values occupy
- 02
The seven ionisable side chains and the pH at which each switches
- 03
Histidine as the only residue that changes state near physiological pH
- 04
Reading a pK from a titration step by two independent methods
- 05
Normalising a titration to moles of base per mole of acid
- 06
Isoelectric point as the average of the two bracketing pK values
- 07
Net charge as a prediction about a peptide at a stated pH
Net charge of a short peptide at two pH values
- 2List every ionisable group with its pK and compare each with the working pH.
- 2Assign a charge to each group at pH 7.0 and total them.
- 2Repeat at pH 2.0 and name which groups changed.
Key terms
- Zwitterion
- A molecule carrying both a positive and a negative charge at the same time with no net charge overall, which is the state of a free amino acid backbone near neutral pH.
- Side-chain pK
- The pK of the ionisable group on a residue's side chain, listed separately from the two backbone values because it is the value that varies between residues.
- Isoelectric point
- The pH at which a molecule carries no net charge, obtained by averaging the two pK values that bracket the electrically neutral species.
- Imidazole group
- The side chain of histidine, whose pK lies close to physiological pH so that both the protonated and unprotonated forms are present in useful amounts inside a cell.
- Point of inflexion
- The centre of symmetry of a titration step, where the curve changes direction. Reading a pK there avoids relying on the accuracy of the titrant concentration.
- Equivalent
- One mole of titratable protons per mole of the substance being titrated, used as the horizontal axis so that curves become independent of the volumes and concentrations used.
Amino Acids and Their Ionisation FAQ
Why is histidine used for the titration in the first practical?
Because it is the only common residue whose side chain changes protonation state near the pH at which cells operate, with the imidazole pK falling in the range six to seven depending on temperature, ionic strength and neighbouring groups. That makes it both a useful biological buffer and a clear teaching example, since its curve shows three separate steps within an accessible range.
How do I decide which two pK values to average for the isoelectric point?
Write the molecule out as the pH rises, from fully protonated through to fully deprotonated, and identify the species whose positive and negative charges cancel. The two pK values immediately above and below that species are the ones to average. Averaging all three, or picking the two largest, gives an answer that is wrong for every residue with a charged side chain.
Why plot against moles of base per mole of acid instead of volume added?
Because that normalisation removes the volume and the concentrations from the plot, so the shape no longer depends on how the experiment happened to be scaled. Midpoints then fall at half an equivalent, one and a half equivalents and so on, and two groups with different starting volumes produce curves that can be compared directly.
Exam move
Memorise the seven side-chain pK values as three groups rather than seven numbers: the two acidic residues near four, histidine near six, and the remaining four above eight. Then practise the charge question in both directions, predicting charge from a pH and predicting a pH range from a stated charge. Sketch the histidine curve from memory at least once before the first assessment.
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