Amino acids, zwitterions and the isoelectric point

An egg, a hair and a muscle

Egg white, hair, muscle and the enzymes in your stomach are all proteins. A protein is a long chain of smaller molecules called amino acids. About twenty kinds of amino acid are used to build almost every protein in living things. We start with the amino acids, because their chemistry decides what the chain can do.

What an amino acid is

An α-amino acid has an amino group and a carboxyl group on the same carbon atom. This carbon is the α carbon. It also carries a hydrogen atom and a side chain, R:

$\text{H}_{2}\text{N}\text{-}\text{CH}(\text{R})\text{-}\text{COOH}$

The side chain is the only part that changes. In glycine R is a hydrogen atom. In alanine it is $\text{CH}_{3}$. In serine it is $\text{CH}_{2}\text{OH}$. In phenylalanine it is a $\text{CH}_{2}$ group joined to a benzene ring.

We group the 20 standard amino acids by the side chain:

• Non-polar: a hydrocarbon or sulphur ether side chain. Glycine, alanine, valine, leucine, isoleucine, methionine and proline.
• Aromatic: a benzene or indole ring. Phenylalanine, tyrosine and tryptophan.
• Polar, uncharged: an $\text{-}\text{OH}$, $\text{-}\text{SH}$ or amide group. Serine, threonine, cysteine, asparagine and glutamine.
• Acidic: a second $\text{-}\text{COOH}$ group. Aspartic acid and glutamic acid.
• Basic: an extra amino or similar group. Lysine, arginine and histidine.

Essential amino acids. The body cannot make some amino acids, and they must come from food. For an adult, nine are essential: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine. NCERT also lists arginine as essential. Arginine is made by the body, but not in enough quantity for a growing child, so many books call it conditionally essential. The others are non-essential.

Handedness

Every α carbon except the one in glycine has four different groups. It is a stereocentre. Glycine has two hydrogen atoms on its α carbon, so it is achiral.

Natural proteins contain only L amino acids. In the Fischer projection, with the $\text{-}\text{COOH}$ group at the top, the $\text{-}\text{NH}_{2}$ group of an L amino acid is on the left. This matches the L sugars. By the R and S rules, nearly all L amino acids are S. The exception is cysteine, which is R, because the $\text{-}\text{CH}_{2}\text{SH}$ group has a higher priority than the $\text{-}\text{COOH}$ group, since sulphur outranks oxygen.

Isoleucine and threonine have a second stereocentre in the side chain. Each has $2^2 = 4$ stereoisomers.

The zwitterion

An amino acid has a group that gives a proton ($\text{-}\text{COOH}$) and a group that takes one ($\text{-}\text{NH}_{2}$). Inside the same molecule, the acid group passes its proton to the amine. This gives

$\text{H}_{3}\text{N}^{+}\text{-}\text{CH}(\text{R})\text{-}\text{COO}^{-}$

This is a zwitterion, a molecule with a positive and a negative charge and no net charge. In the solid and in water near neutral pH, amino acids are zwitterions, not the uncharged form. That explains their properties: they are crystalline solids with high melting points, they dissolve in water, and they do not dissolve in non-polar solvents. Glycine decomposes near 233 °C before it melts.

Charge and pH

The charge of an amino acid depends on the pH. We take glycine as the example.

• In strong acid, both groups are protonated: $\text{H}_{3}\text{N}^{+}\text{-}\text{CH}_{2}\text{-}\text{COOH}$. The charge is +1.
• In the middle, the carboxyl group has lost its proton: $\text{H}_{3}\text{N}^{+}\text{-}\text{CH}_{2}\text{-}\text{COO}^{-}$. The charge is 0.
• In strong base, the ammonium group has lost its proton too: $\text{H}_{2}\text{N}\text{-}\text{CH}_{2}\text{-}\text{COO}^{-}$. The charge is -1.

Each step has its own pK. For glycine, $\text{p}K_1 = 2.34$ for the $\text{-}\text{COOH}$ group and $\text{p}K_2 = 9.60$ for the $\text{-}\text{NH}_{3}^{+}$ group. At $\text{pH} = \text{p}K$, half of the groups are protonated. Below the pK, most are protonated. Above it, most are not. The Henderson and Hasselbalch relation is

The titration curve of glycine shows both steps.

The pH changes slowly near a pK, where the amino acid acts as a buffer. It jumps in the middle, where one hydroxide has been added for each glycine.

The isoelectric point

The pH at which the average charge of an amino acid is zero is its isoelectric point, pI. For an amino acid with no ionisable side chain, the neutral zwitterion is the main form between the two pK values, so

For glycine this is $(2.34 + 9.60)/2 = 5.97$.

If the side chain also ionises, we use the two pK values on either side of the neutral form:

• For an acidic amino acid, the neutral form lies between the first two pK values. So pI is the mean of the α-COOH and the side-chain pK. For aspartic acid, $(1.88 + 3.65)/2 = 2.77$.
• For a basic amino acid, the neutral form lies between the last two pK values. For lysine, the group with the lower pK, the α-NH3+ group, loses its proton first, at 8.95. The side-chain amine goes next, at 10.53. So $\text{pI} = (8.95 + 10.53)/2 = 9.74$.

• Glycine: pK1 2.34, pK2 9.60, no side-chain pK, pI 5.97.
• Valine: pK1 2.32, pK2 9.62, no side-chain pK, pI 5.97.
• Aspartic acid: pK1 1.88, pK2 9.60, side chain 3.65, pI 2.77.
• Glutamic acid: pK1 2.19, pK2 9.67, side chain 4.25, pI 3.22.
• Lysine: pK1 2.18, pK2 8.95, side chain 10.53, pI 9.74.
• Histidine: pK1 1.82, pK2 9.17, side chain 6.00, pI 7.59.

Here pK1 belongs to the α-COOH group and pK2 to the α-NH3+ group.

The values are the standard ones from biochemistry texts. Textbooks round the pI slightly differently, so you may see a difference of 0.01.

Set pH to 5.97 and read the average charge. It is almost zero. Now move pK2 to 10.2. The isoelectric point shifts to the middle of the new pair.

Practise with new numbers

Try it in Python

The program finds the pI of six amino acids from their pK values and says which electrode each one moves to in a solution at pH 7.

Moving in an electric field

Place an amino acid in a solution with two electrodes. If the pH is above its pI, the molecule is mostly negative and moves to the positive electrode, the anode. If the pH is below its pI, it is mostly positive and moves to the negative electrode, the cathode. At the pI it does not move. This is electrophoresis. It can separate amino acids and proteins.

Tests and reactions of amino acids

• Ninhydrin. Warm an α-amino acid with ninhydrin and a purple colour appears. Proline gives a yellow colour, because its nitrogen is a secondary amine. The test finds amino acids and peptides with a free amino group.
• Nitrous acid. A free primary amino group gives off nitrogen gas: $\text{R}\text{-}\text{NH}_{2} + \text{HNO}_{2} \to \text{R}\text{-}\text{OH} + \text{N}_{2} + \text{H}_{2}\text{O}$. Proline does not react.
• Esterification. The carboxyl group reacts with an alcohol and an acid catalyst to give an ester.
• Acylation. The amino group reacts with an acid chloride or anhydride to give an amide.
• Peptide bond. The amino group of one amino acid joins to the carboxyl group of another. That is the subject of the next lesson.

Common mistakes

• Writing the uncharged form $\text{H}_{2}\text{N}\text{-}\text{CH}(\text{R})\text{-}\text{COOH}$ for a solid amino acid. In the solid and near neutral pH it is a zwitterion.
• Averaging the wrong two pK values. For an acidic amino acid, use the carboxyl groups. For a basic one, use the two amino groups.
• Thinking the pI is where the amino acid is most soluble. It is usually where it is least soluble, because there is no net charge to hold the molecules apart.
• Saying that all L amino acids are R or all are S. Nearly all are S, but cysteine is R.

Remember these

• An α-amino acid is $\text{H}_{2}\text{N}\text{-}\text{CH}(\text{R})\text{-}\text{COOH}$. Natural proteins have L amino acids only. Glycine is achiral.
• In water an amino acid is a zwitterion. Its charge is $+1$ at low pH, $0$ at the pI and $-1$ at high pH.
• For a neutral side chain, $\text{pI} = (\text{p}K_1 + \text{p}K_2)/2$. For acidic or basic side chains, average the two pK values on either side of the neutral form.
• Above the pI, a molecule moves to the anode. Below the pI, it moves to the cathode.
• Ninhydrin gives purple (proline yellow). Nitrous acid releases $\text{N}_{2}$.

Check yourself

Try each one on your own first. If you are stuck, open a hint. Then check with Show the answer.

1. Write the form of alanine that predominates at pH 1.

2. Find the pI of an amino acid with an acidic side chain, given $\text{p}K_1 = 2.0$, $\text{p}K_2 = 9.8$ and $\text{p}K_R = 4.1$.

3. At pH 8, will an amino acid with pI 9.74 move to the anode or the cathode?

4. Why do amino acids have much higher melting points than amines or carboxylic acids of similar size?

References

• Nelson and Cox, Lehninger Principles of Biochemistry, 8th edition, Chapter 3 (amino acids, Table 3-1 for the pK values).
• NCERT Chemistry for Class 12, Part II, the chapter Biomolecules (amino acids, zwitterion).
• OpenStax, Organic Chemistry, Chapter 26 (Biomolecules: Amino Acids, Peptides and Proteins).