Biochemistry – Amino Acids, Peptides & Proteins (BCHEM 154)

Flashcards cover core concepts from the lecture transcript, including amino-acid chemistry, properties, reactions, peptide sequencing methods, protein structure, denaturation, and common purification techniques.

What four atoms/groups are bonded to the α-carbon of a standard amino acid?

An amino group, a carboxyl group, a hydrogen atom, and a distinctive side-chain (R group).

Which amino acid lacks chirality and why?

Glycine, because its R group is a hydrogen atom, giving the α-carbon two identical substituents.

At physiological pH, what ionic form do most amino acids adopt?

A zwitterion with a deprotonated carboxylate (–COO⁻) and a protonated amino group (–NH₃⁺).

What is the general pKa range for α-carboxyl groups of amino acids?

Approximately 2.0.

What is the general pKa range for α-amino groups of amino acids?

Between 9.0 and 10.5.

Name the two acidic, negatively charged amino acids at pH 7.

Aspartic acid (Asp, D) and glutamic acid (Glu, E).

Which three amino acids have basic, positively charged side chains at neutral pH?

Lysine (Lys, K), arginine (Arg, R) and histidine (His, H).

What unique ring structure is found in tryptophan?

An indole ring.

Why is proline considered an imino acid?

Its side-chain forms a cyclic bond with the α-amino nitrogen, giving it a secondary (imino) amino group.

Define ‘essential amino acids’.

Amino acids that cannot be synthesised by higher animals and must be supplied in the diet.

Give any four essential amino acids for humans.

Examples: Lysine, leucine, isoleucine, valine, methionine, phenylalanine, threonine, tryptophan, histidine (in children).

What is transamination?

Transfer of an amino group from an amino acid to an α-keto acid, forming a new amino acid and a new keto acid, catalysed by transaminases.

Which co-enzyme is commonly required for transaminase enzymes?

Pyridoxal phosphate (vitamin B₆ derivative).

Write the Henderson–Hasselbalch equation.

pH = pKa + log([A⁻] / [HA]).

At pH = pKa, what fraction of an ionisable group is deprotonated?

50 % (the group is half-dissociated).

How is the isoelectric point (pI) of a neutral amino acid calculated?

pI = (pKa₁ + pKa₂) ⁄ 2, where pKa₁ is for –COOH and pKa₂ for –NH₃⁺.

Describe the ninhydrin reaction outcome for most amino acids.

They yield Ruhemann’s purple, an intense violet-blue colour, allowing quantitative detection.

Which amino acids give a yellow colour with ninhydrin?

Proline and hydroxyproline (imino acids).

What reagent is used in Sanger’s method for N-terminal analysis?

2,4-dinitrofluorobenzene (DNFB).

What is the Edman degradation reagent?

Phenylisothiocyanate (PITC).

State one advantage of dansyl chloride over Sanger’s reagent.

Much greater sensitivity; fluorescence allows detection of minute amounts of the N-terminal amino acid.

What is a peptide bond chemically?

An amide linkage between the α-carboxyl group of one amino acid and the α-amino group of another, releasing water.

Name any two naturally occurring small peptides and a key function of each.

Oxytocin – uterine contraction; Vasopressin (ADH) – water reabsorption & vasoconstriction.

What is meant by peptide bond ‘partial double-bond character’?

Resonance gives the C–N bond partial double-bond qualities, making it planar and restricting rotation.

Differentiate fibrous and globular proteins in terms of solubility.

Fibrous proteins are generally water-insoluble; globular proteins are usually water-soluble.

Give two examples of fibrous proteins and their roles.

Collagen – connective tissue strength; keratin – hair and nails structure.

What are the four levels of protein structure?

Primary, secondary, tertiary, and quaternary structures.

Name two common secondary structural elements.

α-helix and β-pleated sheet.

What stabilises an α-helix?

Intramolecular hydrogen bonds between carbonyl oxygen of residue i and amide hydrogen of residue i+4.

Contrast parallel and antiparallel β-sheets.

Parallel strands run in the same N→C direction; antiparallel strands run in opposite directions, affecting H-bond geometry.

Which amino acid frequently introduces kinks and disrupts α-helices?

Proline.

Define protein denaturation.

Loss of native 3-D conformation (and usually function) due to disruption of non-covalent interactions or disulfide bonds.

List three physical agents that cause protein denaturation.

Heat, high pressure, vigorous shaking or foaming, freezing.

How do urea and detergents denature proteins?

They disrupt hydrogen bonds and hydrophobic interactions, respectively.

What reducing agent is commonly used to break disulfide bonds during denaturation?

β-mercaptoethanol (2-ME) or dithiothreitol (DTT).

Explain ‘salting-in’ and ‘salting-out’.

Low salt increases protein solubility (salting-in); high salt competes for water and precipitates proteins (salting-out).

Why are proteins least soluble at their isoelectric point?

Net charge is zero, so electrostatic repulsion is minimal and molecules aggregate via van der Waals and hydrophobic interactions.

Name two common salts used for salting-out proteins.

Ammonium sulfate and sodium sulfate.

What is the Biuret test specific for?

Peptide bonds (proteins with at least two peptide linkages) producing a purple complex with Cu²⁺ in alkaline solution.

Which amino acid side chain gives a positive xanthoproteic test?

Aromatic side chains (phenylalanine, tyrosine, tryptophan).

What functional group in arginine yields the red Sakaguchi reaction?

Guanidinium group.

Which amino acid side chain forms disulfide bonds?

Cysteine (through its –SH group).

Describe the role of histidine in metal-binding proteins.

Its imidazole nitrogen donates a lone pair to coordinate metal ions (e.g., Fe²⁺ in haemoglobin, Cu²⁺ in cytochrome c).

Which reagent selectively cleaves peptide bonds on the C-side of methionine residues?

Cyanogen bromide (CNBr).

Which protease cleaves after lysine and arginine?

Trypsin.

Name the bacterial enzyme that preferentially hydrolyses peptide bonds on the N-side of bulky hydrophobic residues.

Thermolysin.

Why is overlapping peptide strategy necessary in sequencing?

To align fragments from different cleavage methods and reconstruct the entire primary sequence.

Define a protein’s ‘domain’.

A compact, independently folded region of tertiary structure often associated with a specific function.

What is a ‘prosthetic group’?

A non-protein component tightly bound to a conjugated protein essential for its function, e.g., heme in hemoglobin.

Differentiate simple and conjugated proteins.

Simple proteins consist only of amino acids; conjugated proteins contain amino acids plus a prosthetic group.

Explain the principle of ion-exchange chromatography for amino acids.

Amino acids bind to oppositely charged resin; elution with varying pH or ionic strength separates them according to net charge.

What detection method follows ion-exchange separation in an amino acid analyser?

Post-column reaction with ninhydrin and absorbance measurement at 570 nm (440 nm for proline/hydroxyproline).

In SDS-PAGE, what does SDS do to proteins?

Denatures proteins and imparts a uniform negative charge proportional to length, allowing size-based separation.

State the sedimentation coefficient unit and what it measures.

The Svedberg (S); it measures a particle’s sedimentation rate during ultracentrifugation, reflecting size and shape.

Give the equation relating electrophoretic velocity (v) to electric field strength (E).

v = (E × z) ⁄ f, where z is net charge and f is frictional coefficient.

What is affinity chromatography based on?

Specific reversible binding between a protein and a ligand immobilised on the column matrix.

How is protein purity often verified after each purification step?

By measuring specific activity (activity per milligram protein) or observing a single band in SDS-PAGE/electrophoresis.

Why does HbS polymerise under low O₂ tension?

Valine substitution at position 6 in β-chain introduces a hydrophobic patch, promoting intermolecular hydrophobic interactions.

List two consequences of sickle-cell haemoglobin polymerisation.

Erythrocyte deformation into sickle shape and obstruction of capillaries leading to tissue hypoxia and pain.

Which rare amino acid is abundant in collagen and what is its origin?

4-Hydroxyproline, a post-translational derivative of proline.

What is the significance of primary structure to tertiary structure?

Primary sequence dictates folding pattern and final 3-D conformation, and thus biological function.

Describe the principle of gel-filtration chromatography.

Porous beads sieve molecules; large proteins elute first as they do not enter pores, small ones elute later.