Proteins are naturally occurring macromolecules consisting of polypeptide chains folded into precise 3D configurations. They are held together by peptide linkages formed between amino acid monomer units.
🔑 Key Principle
A peptide link (or peptide bond) is formed by a condensation reaction between the carboxylic acid group of one amino acid and the amine group of another, releasing one water molecule.
The covalent amide link (\\( \text{-CONH-} \\)) formed between the carboxyl carbon of one amino acid and the amino nitrogen of another.
Dipeptide Formation
When two amino acids combine, they form a dipeptide. If three combine, they form a tripeptide. Polypeptides are longer chains, containing many repeating amino acid residues.
Dipeptide Isomers
When reacting two different amino acids (e.g. Alanine and Glycine), two different dipeptide structural isomers can form depending on which amino acid donates the amine group and which donates the carboxylic acid group:
- Ala-Gly (Alanine N-terminus, Glycine C-terminus)
- Gly-Ala (Glycine N-terminus, Alanine C-terminus)
Levels of Protein Structure
Proteins possess four levels of structural organization, though the AQA specification focuses on the first three:
Primary Structure: The unique sequence of amino acids in a polypeptide chain.
Secondary Structure: The local folding of the polypeptide chain into repeating shapes (such as an alpha-helix or beta-pleated sheet), stabilized by hydrogen bonds between peptide groups.
Tertiary Structure: The complex 3D folding of a single polypeptide chain, stabilized by interactions between amino acid R group side chains.
Stabilisation of Tertiary Structure
Four distinct chemical interactions between amino acid side chains (R groups) stabilize the overall 3D tertiary conformation:
- Hydrogen Bonds: Form between polar R groups (e.g. groups containing \\( \text{-OH} \\) or \\( \text{-NH}_2 \\)). They are weak but numerous.
- Ionic Interactions (Salt Bridges): Form between charged R groups (e.g. \\( \text{-COO}^- \\) and \\( \text{-NH}_3^+ \\) side chains).
- van der Waals Forces (Induced Dipole-Dipole): Form between non-polar hydrophobic R groups (e.g. alkyl chains of alanine or leucine).
- Disulfide Bridges: Covalent bonds that form when the sulfur atoms of two adjacent cysteine amino acids are oxidized. These are very strong covalent bonds.
A strong covalent bond formed by the oxidation of two thiol (\\( \text{-SH} \\)) groups on adjacent cysteine amino acid residues, yielding a \\( \text{-S-S-} \\) linkage.
Protein Hydrolysis
Polypeptides and proteins can be broken back down into their component amino acids through hydrolysis. This is the reverse of condensation:
- Reagents and Conditions: Heating the protein with \\( 6 \text{ mol dm}^{-3} \text{ HCl} \\) at reflux for 24 hours.
- Products: The peptide links cleave, yielding protonated amino acids (cations) because the reaction occurs under strongly acidic conditions.
Chromatographic Analysis of Amino Acids
Once a protein has been hydrolysed, the mixture of resulting amino acids can be separated and identified using Thin-Layer Chromatography (TLC):
- Application: A small spot of the hydrolysed amino acid mixture (and known reference samples) is placed on a TLC plate (stationary phase).
- Development: The plate is placed in a solvent (mobile phase). Different amino acids ascend the plate at different speeds depending on their relative affinity for the stationary phase versus their solubility in the mobile phase.
- Visualisation: Amino acids are colorless. To make the spots visible, the dried plate must be sprayed with a locating agent such as ninhydrin (which reacts with amino acids to form purple spots) or viewed under ultraviolet (UV) light.
- Identification: The distance traveled by each amino acid relative to the solvent front is measured to calculate its retardation factor (\\( R_f \\) value): \[ R_f = \frac{\text{Distance travelled by amino acid}}{\text{Distance travelled by solvent front}} \] Comparing calculated \\( R_f \\) values to database standards allows each amino acid to be identified.
When describing TLC experiments in exams, remember these key practical details:
- Draw the start line in pencil, not ink (ink contains dyes that will dissolve in the solvent and smear up the plate).
- Ensure the level of the solvent in the beaker is below the pencil line so the amino acid spots do not dissolve directly into the solvent reservoir.
- Cover the beaker with a lid to saturate the internal atmosphere with solvent vapour, preventing evaporation off the plate.
- Glycine: \\( R_f = 0.30 \\)
- Alanine: \\( R_f = 0.65 \\)
- Valine: \\( R_f = 0.82 \\)
Step 1: Calculate Rf for Spot A. \[ R_f(A) = \frac{2.4\text{ cm}}{8.0\text{ cm}} = 0.30 \]
Step 2: Calculate Rf for Spot B. \[ R_f(B) = \frac{5.2\text{ cm}}{8.0\text{ cm}} = 0.65 \]
Step 3: Compare with database values.
- Spot A matches Glycine (\\( R_f = 0.30 \\)).
- Spot B matches Alanine (\\( R_f = 0.65 \\)).
Answer: The two amino acids in the dipeptide are glycine and alanine.
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