Proteins (A-level only) — AQA A-Level Chemistry
Test yourself on Proteins (A-level only) with AQA A-Level practice questions.
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Proteins (A-level only) explained
Two amino acids join by condensation: the -COOH of one reacts with the -NH₂ of the next, a molecule of water is lost, and what is left is -CO-NH-, the peptide link, which is an amide group.
Read the full explanation
A protein is that reaction repeated hundreds of times, so the chain has a free amine at one end and a free carboxyl at the other, giving it a direction. Order matters: glycine joined to alanine is a different dipeptide from alanine joined to glycine, because the peptide link points the other way. Three amino acids give a tripeptide with two links, and in general a chain of n residues contains n − 1 peptide links and lost n − 1 molecules of water when it formed.
The importance of hydrogen bonding and sulfur–sulfur bonds in proteins.
Two very different attractions hold a protein in shape. A hydrogen bond forms between the δ⁺ hydrogen of an N-H and a lone pair on the δ⁻ oxygen of a C=O, or between R groups that carry -OH, -NH₂ or -COOH, as a serine R group does with an aspartic acid R group. Each one is weak, but a protein has thousands and together they hold the helix, the sheet and much of the fold. A disulfide bridge is different in kind: two cysteine R groups, each ending in -CH₂SH, are oxidised so their sulfur atoms bond directly, giving -CH₂-S-S-CH₂-. That is a covalent bond, so it is far stronger than a hydrogen bond and pins together parts of the chain that are far apart in the sequence.
The primary, secondary (α-helix and β–pleated sheets) and tertiary structure of proteins.
Primary structure is the sequence of amino acids in the chain, held by the covalent peptide links; it is the one level that hydrolysis has to break. Secondary structure is the regular local shape produced by hydrogen bonds between the C=O of one peptide link and the N-H of another along the backbone: in the α-helix the chain coils and the hydrogen bonds run parallel to the axis, and in the β-pleated sheet lengths of chain lie side by side with hydrogen bonds bridging between them. Tertiary structure is the way the whole chain, helices and sheets together, folds into one three-dimensional shape, held by hydrogen bonds, ionic attractions between charged R groups, van der Waals forces and disulfide bridges.
Hydrolysis of the peptide link produces the constituent amino acids.
Hydrolysis is condensation run backwards: a molecule of water is put back across every peptide link, cutting the -CO-NH- bond and returning the individual amino acids. It is slow, so the protein is heated under reflux for several hours with about 6 mol dm⁻³ hydrochloric acid. The conditions change the form the products take. In that excess of acid every amine group is protonated, so the amino acids come out as their cations. Hydrolysis with hot aqueous alkali instead gives the carboxylate salts and free -NH₂ groups. A chain of n residues gives n amino acids and consumes n − 1 molecules of water. The mixture is then usually separated and identified by thin-layer chromatography.
Amino acids can be separated and identified by thin-layer chromatography.
Thin-layer chromatography separates amino acid mixtures between two phases. The stationary phase is silica or alumina; the mobile phase is a solvent moving up by capillary action. Samples are spotted on a pencil baseline. The plate stands in a sealed tank with solvent below the line. A lid prevents solvent evaporation. Amino acids separate based on their relative solubility in the mobile phase and adsorption to the stationary phase. Because amino acids are colourless, a chemical locating agent like ninhydrin is sprayed onto the dried plate to make spots visible. Identification is achieved by calculating the Rf value (distance moved by spot divided by distance moved by solvent front) and comparing it to known standard values.
Amino acids can be located on a chromatogram using developing agents such as ninhydrin or ultraviolet light and identified by their R values.
Amino acids are colourless, so a finished plate looks blank and the spots have to be made visible. Spraying with ninhydrin turns each spot purple; iodine also works, and a plate made with a fluorescent additive shows the spots as dark patches under an ultraviolet lamp. Once the spots are visible, the positions are measured. The Rf value is the distance travelled by the spot divided by the distance travelled by the solvent front, both measured from the pencil baseline and the spot measured to its centre. Rf always lies between 0 and 1. It is only fixed for a given solvent and stationary phase, so a known sample is run alongside on the same plate for comparison.
Students should be able to: draw the structure of a peptide formed from up to three amino acids draw the structure of the amino acids formed by hydrolysis of a peptide identify primary, secondary and tertiary structures in diagrams explain how these structures are maintained by hydrogen bonding and S–S bonds calculate Rf values from a chromatogram.
This pulls the section's skills together. Building a peptide: join the -COOH of one residue to the -NH₂ of the next, remove a water molecule at each junction and keep the residues in the stated order. Taking one apart: cut every C-N bond of a link, give the carbonyl carbon an -OH and the nitrogen an -H, and adjust for acid or alkaline conditions. Reading a diagram: peptide links mean primary structure, backbone hydrogen bonds mean an α-helix or a β-pleated sheet, and R group interactions, including disulfide bridges between cysteine residues, mean tertiary structure. Reading a chromatogram: divide the distance the spot moved by the distance the solvent front moved, both from the pencil line.
Your focus
- Draw the structure of a dipeptide or tripeptide formed from named amino acids in a stated order.
- Complete a peptide structure that is only partly drawn, putting the hydrogen on the nitrogen of every peptide link and the right group - acid or amine - at the end of the chain.
- Calculate how many molecules of water are released when a chain of a given length forms.
Show all 21 objectives
- Identify from a pair of R groups which interaction, hydrogen bonding or a disulfide bridge, can form between them.
- Explain why a disulfide bridge is a stronger interaction than a hydrogen bond.
- Draw a hydrogen bond between two named R groups, showing the lone pair and the partial charges.
- Define primary, secondary and tertiary structure and state the bond or force that maintains each.
- Identify the α-helix, the β-pleated sheet and the tertiary fold in a labelled protein diagram.
- Explain why breaking hydrogen bonds changes a protein's shape without changing its primary structure.
- Draw the amino acids released when a given di- or tripeptide is hydrolysed, in the form the stated conditions give.
- State the reagent, concentration and conditions used to hydrolyse a protein completely.
- Deduce the number of water molecules consumed when a peptide of a stated length is hydrolysed.
- Describe the correct setup of a TLC tank, including a pencil baseline above the solvent and a lid to saturate the atmosphere.
- Explain how amino acids are visualised using a chemical locating agent such as ninhydrin.
- Calculate Rf values from a chromatogram and use them to identify amino acids by comparison with known standards.
- Name a suitable developing agent for amino acids and state why one is required.
- Calculate an Rf value from measurements taken off a chromatogram.
- Explain why an unknown must be run alongside a known standard on the same plate before it can be identified.
- Deduce how many spots a chromatogram of a fully hydrolysed peptide should show when two of its amino acids are the same, and say what each spot represents.
- Identify where the hydrogen bonds of an α-helix or β-pleated sheet run - between the N-H and C=O of the backbone, not between R groups - and use that to tell secondary from tertiary structure in a diagram.
- Calculate an Rf value from a chromatogram and use it with a table of standards to identify an amino acid.
Proteins (A-level only) exam tips
Marking Points
- One mark for the peptide link drawn as a C=O joined directly to an N-H.
- One mark for stating one molecule of water is lost for each peptide link formed.
- One mark for explicitly showing the hydrogen on the nitrogen of the peptide link in both displayed and skeletal structures.
- One mark for building the amino acid residues in the exact order the sequence specifies.
- one mark for naming cysteine as the amino acid whose R groups form the disulfide bridge
- one mark for hydrogen bonding between R groups such as those of serine and aspartic acid
- one mark for stating that the disulfide bridge is the stronger interaction
- one mark for the reason, that a disulfide bridge is a covalent bond whereas a hydrogen bond is not
- one mark for primary structure defined as the sequence of amino acids joined by peptide links
- one mark for secondary structure held by hydrogen bonds between the C=O and N-H of the backbone
- one mark for naming the α-helix and the β-pleated sheet as the two secondary structures
- one mark for tertiary structure as the overall folding, held by interactions between R groups including S-S bridges
- one mark for heating under reflux with about 6 mol dm⁻³ hydrochloric acid for several hours
- one mark for one molecule of water added for each peptide link broken
- one mark for the amino acids drawn as their protonated cations under acidic conditions
- one mark for all the residues in the chain being released, not only one or two
- State that separation depends on the balance between solubility in the mobile phase and retention (adsorption) on the stationary phase.
- Identify that a pencil baseline must be drawn above the solvent level to prevent spots dissolving into the reservoir.
- Explain that a chemical locating agent, such as ninhydrin, is required to visualise colourless amino acid spots.
- Describe how to identify an amino acid by calculating its Rf value and comparing it against a database of known standard values.
- one mark for naming ninhydrin, or iodine, as the developing agent
- one mark for the reason it is needed, that the amino acids are colourless
- one mark for the Rf calculated as the distance moved by the spot divided by the distance moved by the solvent front
- one mark for both distances measured from the pencil baseline, with the spot measured to its centre
- one mark for each peptide link drawn correctly when a di- or tripeptide is built
- one mark for the correct amino acids obtained by cutting every link and adding water back
- one mark for naming the bond or force that maintains a labelled feature in a protein diagram
- one mark for an Rf value calculated as spot distance over solvent front distance from the baseline
Examiner Tips
- 💡Write the sequence out left to right with the amine end first, then join them in that order to avoid reversing the peptide link.
- 💡In both displayed and skeletal formulae, the H on the peptide nitrogen must be explicitly drawn because hydrogens attached to heteroatoms (like N or O) cannot be omitted.
- 💡Use the data booklet to read the R groups before you decide what interaction is possible; the R group is what the mark depends on.
- 💡A diagram showing at least -S-S- can earn the disulfide mark on its own.
- 💡Identify the level first from what is holding it: peptide links means primary, backbone hydrogen bonds means secondary, R group interactions means tertiary.
- 💡In a diagram question, point at the feature and name both the level and the force that maintains it.
- 💡Mark the C-N bond of each link with a line before you cut; that stops you splitting the molecule in the wrong place.
- 💡Read the conditions in the stem: acid gives cations, alkali gives anions, and the neutral form is only right if the question says so.
- 💡Always name both phases in your explanation: mention solubility in the mobile phase and adsorption to the stationary phase.
- 💡When asked how to identify the separated amino acids, explicitly state the formula for Rf value and mention comparing it to known standards.
- 💡State the final colour only as extra detail; on its own 'it turns them purple' does not explain why a developing agent is needed.
- 💡Show the two distances in your working before you divide, so a method mark survives an arithmetic slip.
- 💡The specification mentions ultraviolet light, but when AQA asks you to NAME a developing agent the mark scheme ignores UV: write ninhydrin (or iodine).
- 💡Draw the backbone first, N to C, then hang the R groups on the α-carbons; most structure marks are lost on the skeleton, not the side chains.
- 💡Show the division as a fraction with both measured distances before you evaluate it.
Common Mistakes
- Joining two -NH₂ groups, or two -COOH groups, to each other; correct by ensuring the -COOH of one amino acid reacts with the -NH₂ of the adjacent amino acid.
- Leaving the hydrogen off the nitrogen of a peptide link in a skeletal formula; correct by always drawing the H attached to the N atom, as hydrogens on heteroatoms must be shown.
- Treating a dipeptide and its reverse as the same compound; correct by recognising that sequence direction matters (e.g., Gly-Ala is structurally different to Ala-Gly).
- Keeping the -OH on the carbonyl carbon so no water has actually been lost; correct by removing the -OH from the carboxyl group and one -H from the amine group.
- calling a disulfide bridge a hydrogen bond because it holds two parts of a chain together
- drawing a hydrogen bond to the hydrogen atom of a C=O rather than to a lone pair on the oxygen
- describing hydrogen bonds as covalent
- claiming any pair of R groups can form an S-S bridge, when only cysteine has the -SH group
- saying the secondary structure is held together by peptide links
- placing disulfide bridges in the secondary structure rather than the tertiary
- describing tertiary structure as just the shape of the helix
- confusing the primary structure with the molecular formula of the protein
- drawing the neutral amino acid as the product of hydrolysis in excess hydrochloric acid
- drawing only one of the fragments when the peptide contained three residues
- adding water across the C=O bond rather than across the C-N bond of the link
- assuming the R groups are altered or lost during hydrolysis
- Drawing the baseline in ink, which separates and obscures the amino acids; correct by specifying a pencil baseline.
- Stating UV light is a locating agent for amino acids; correct by explicitly stating a chemical locating agent like ninhydrin is needed, as UV light is a physical visualisation method.
- Explaining separation by solubility alone; correct by always referencing both solubility in the mobile phase and adsorption to the stationary phase.
- measuring the solvent front from the bottom edge of the plate rather than from the baseline
- measuring to the leading edge of a spot instead of its centre
- quoting an Rf value greater than 1, which means the two distances have been divided the wrong way round
- treating an Rf value as a fixed property of a substance regardless of the solvent used
- building a tripeptide with an R group attached to a carbonyl carbon instead of the α-carbon
- cutting the chain at a C-C bond rather than at the C-N bond of the peptide link
- labelling an α-helix as tertiary structure
- dividing the solvent front distance by the spot distance, giving an Rf greater than 1