Amino Acids and Peptides: Zwitterions and the Peptide Bond
Amino acids contain acidic and basic groups whose protonation changes with pH. Ionisable side chains can add charge, so the whole molecule need not be neutral near pH 7. Peptide bonds join residues through nearly planar amide groups with restricted rotation.
What a learner can do afterwards
- Draws the dominant form of an amino acid at a stated pH and calculates its isoelectric point from the pKa values
- Explains why the peptide bond is planar using delocalisation and predicts the preferred geometry
- Explains why peptide synthesis needs both activation and protection, and where each is applied
- Predicts the direction an amino acid moves during electrophoresis at a given pH
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Protein amino acids have an alpha carbon bonded to an amino nitrogen, carboxyl group, hydrogen and side chain. Proline's side chain also bonds to its nitrogen, forming a ring. Near pH 7, alanine mainly has NH3+ and COO- groups, with net charge zero. This form is a zwitterion. Solution equilibria set protonation; a direct internal proton transfer is not required. Ionisable side chains can give other amino acids a nonzero net charge.
Compare pH with each ionisable group's pKa. Below its pKa, the protonated form is favoured; above it, the deprotonated form is favoured. Far below both alanine pKa values, NH3+ and COOH give net charge +1. Far above both, NH2 and COO- give -1. Near a pKa, substantial amounts of both forms occur. Include any ionisable side chain in the charge sum.
The isoelectric point, pI, is the pH where average net charge is zero. In the usual simple amino-acid model, estimate pI by averaging the two pKa values surrounding the zero-charge form. Track charges to choose the pair, including side chains. For illustrative values 2.1, 4.1 and 9.6 with successive charges +1, 0, -1 and -2, use 2.1 and 4.1: pI is about (2.1 + 4.1) / 2 = 3.1.
For glutamic acid, use illustrative pKa values 2.2 (alpha COOH), 4.3 (side-chain COOH) and 9.7 (NH3+). Compare pH with each pKa: above favours deprotonation; below favours protonation. At pH 7, the main form is NH3+-CH(COO-)-CH2-CH2-COO-. Its charges give +1 - 1 - 1 = -1, so it drifts toward the positive anode. The zero-charge form lies between the two carboxyl dissociations; pI is about 3.25.
At pH 7, add the charges in glutamic acid's main form: two carboxylates contribute -2 and the amino group contributes +1.
In electrophoresis, free amino acids drift according to average charge, without requiring their oxidation or reduction. Positive charge gives drift toward the negative cathode; negative charge gives drift toward the positive anode. Below pI average charge is positive; above pI it is negative. Glutamic acid at pH 7 therefore drifts toward the anode. At pI there is no net electrophoretic drift, though diffusion and fluid motion can still move molecules.
A peptide bond joins one residue's carboxyl carbon to the next residue's amino nitrogen. Nitrogen's lone pair delocalises with the carbonyl, giving the C-N bond partial double-bond character. The peptide unit is nearly planar, and C-N rotation is restricted, not impossible. Most peptide bonds favour trans because it reduces steric crowding. Bonds immediately before proline have a larger cis fraction than other peptide bonds.
A free carboxylic acid and amine often form an ammonium carboxylate salt rather than a peptide. For controlled laboratory coupling, activate the carboxyl group, for example as an activated ester, to aid amine attack. Protect groups that could react at unwanted sites, leaving the intended pair available. Coupling forms the amide. Remove selected protecting groups when needed; some remain through several couplings.
Use pH and pKa to track charge and estimate pI. Charge sets electrophoretic drift. Delocalisation restricts peptide rotation; activation and protection control coupling.
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More to read
Identify the backbone before counting charge
An alpha amino acid places its amino nitrogen and carboxyl group at the same central carbon. That carbon also bonds to a hydrogen and a side chain. In glycine, the side chain is another hydrogen. Proline has a ring connecting its side chain to the amino nitrogen, which makes its amino group different from the usual primary amino group.
Group identity and group charge are separate questions. A carboxyl group may be written as COOH or COO-, depending on protonation. An ordinary amino group may be written as NH2 or NH3+.
Near pH 7, alanine mainly has NH3+ and COO- groups. The opposite formal charges cancel, giving a zwitterion. This charge pattern follows equilibria with the solution; it does not establish that a proton travelled directly between the two groups inside one molecule.
Include the side chain in the charge sum
Amino-acid charge calculations must include every group that ionises over the pH range being considered. Counting only the amino and carboxyl groups can miss a side-chain charge.
Glutamic acid has two carboxyl groups. Near pH 7, both are mainly COO-, contributing -2 together. Its amino group is mainly NH3+, contributing +1. The main molecular form therefore has charge -1 even though the backbone contains opposite charges.
Some side chains instead gain a positive charge when protonated. The relevant pKa values determine which forms are abundant. A label such as acidic or basic summarises chemical behaviour; it does not assign one permanent charge at every pH.
Side chains also affect interactions in proteins. Their chemical properties contribute to folding, but the surrounding water, other side chains and peptide backbone all matter. Charge accounting is one part of that chemistry.
Follow successive proton losses
For each ionisable group, compare pH with pKa. A lower pH favours its protonated form. A higher pH favours its deprotonated form. Near pKa, both forms are present in substantial amounts.
Alanine provides a two-group example. At sufficiently low pH, its amino group is NH3+ and its carboxyl group is COOH, giving total charge +1. Loss of the carboxyl proton changes COOH to COO- and lowers the total charge to zero. Loss of an amino-group proton at higher pH changes NH3+ to NH2, giving total charge -1.
These are main forms, not claims that every molecule has the same state. A solution contains an equilibrium mixture. When a side chain also ionises, add the corresponding proton-loss states and keep track of their charges in the same way.
Locate zero average charge
The isoelectric point, pI, is the pH at which average net charge is zero. For the usual simple amino-acid model, an estimate comes from the mean of the two pKa values surrounding the zero-charge form.
Choose this pair by following successive charge states. If an acidic side chain is present, the zero-charge form may lie between two carboxyl dissociations. Averaging the lowest and highest pKa values would then select the wrong interval. Basic side chains can require a different pair at higher pH.
A free amino acid below its pI has positive average charge and drifts toward the negative cathode in an electric field. Above pI it has negative average charge and drifts toward the positive anode.
At pI there is no net electrophoretic drift. Molecules can still diffuse, and flowing liquid can carry them. Zero electric-field drift is not complete immobility.
Control peptide geometry and coupling
A peptide link is an amide joining one residue's carboxyl carbon to another residue's amino nitrogen. Delocalisation of the nitrogen lone pair with the carbonyl gives the C-N bond partial double-bond character. The peptide unit is nearly planar, and rotation about C-N faces a substantial barrier.
Most peptide bonds favour trans geometry because it reduces steric crowding. Cis geometry remains possible. Bonds immediately before proline have a greater cis fraction than other peptide bonds.
Preparing a selected peptide in the laboratory also requires control of reactivity. A free acid and amine often form an ammonium carboxylate salt. Carboxyl activation produces a more reactive partner for amine attack. Protecting groups mask other sites that might react.
The intended coupling groups must be available at the correct stage. Some protecting groups are removed between couplings, while others remain until later. The synthesis determines the schedule.
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