BBCCT 105 Proteins Solved Question Paper June 2026 for IGNOU B.Sc. (Honours) Biochemistry (BSCBCH)

SDS-PAGE and Sanger's method of protein sequencing

Question 3 (a): Explain the principle and applications of SDS-PAGE. (7)

Question 3 (b): Describe Sanger's method of protein sequencing. (7)

(a) Principle and applications of SDS-PAGE

SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) is a denaturing electrophoresis technique that separates polypeptide chains almost entirely on the basis of their molecular mass. It is the most widely used method for checking the size and purity of proteins in a biochemistry laboratory.

Principle. In ordinary electrophoresis a protein moves according to its charge, size and shape together. SDS removes two of those variables. It is an anionic detergent that unfolds the protein and binds along the chain at about 1.4 g of SDS per gram of protein, roughly one SDS molecule for every two amino acid residues.

The bound SDS coats every polypeptide with a large, uniform negative charge, so all SDS-protein complexes have nearly the same charge-to-mass ratio and a similar rod-like shape. A reducing agent such as β-mercaptoethanol or dithiothreitol (DTT) is added and the sample is heated, which breaks disulphide bonds and separates the subunits.

The complexes are then run through a polyacrylamide gel, made by polymerising acrylamide with the cross-linker N,N'-methylenebisacrylamide using ammonium persulphate and TEMED. The gel acts as a molecular sieve: small chains slip through the pores quickly while large chains are held back, so migration depends on size alone.

Most laboratories use the discontinuous Laemmli system (1970). A low-percentage stacking gel at pH 6.8 concentrates the sample into a sharp band, and a resolving gel at pH 8.8, usually 8 to 15 per cent acrylamide, separates the chains in Tris-glycine running buffer at pH 8.3. Over the useful range, relative mobility falls in a straight line as the logarithm of molecular mass rises.

After the run, bands are seen by staining with Coomassie Brilliant Blue R-250 or, for very small amounts, silver stain. Marker proteins of known mass run in a separate lane; a standard curve of log molecular mass against relative mobility (R_f) is drawn and the mass of an unknown band is read from it.

Diagram to draw: a vertical gel slab with the cathode (negative) at the top and anode (positive) at the bottom, labelled stacking gel, resolving gel, sample wells, marker lane and separated bands.

Applications

1. Estimating the molecular mass of polypeptide chains from a marker standard curve.
2. Checking the purity of a protein preparation at each step of purification; a pure single-chain protein gives one band.
3. Finding subunit composition by comparing reduced and non-reduced samples, for example the heavy and light chains of IgG.
4. Western blotting, where separated proteins are transferred to a membrane and detected with specific antibodies.
5. The second dimension of two-dimensional gel electrophoresis in proteomics, after isoelectric focusing.
6. Screening recombinant protein expression and detecting abnormal proteins in clinical and quality-control work.

Because it is quick, inexpensive and needs only micrograms of protein, SDS-PAGE is usually the first analytical check made on any protein sample.

(b) Sanger's method of protein sequencing

Frederick Sanger's method was the first successful approach to working out the complete amino acid sequence of a protein. Using it, he solved bovine insulin, with its A chain of 21 residues and B chain of 30 residues, completing the work in 1955. He received the Nobel Prize in Chemistry in 1958 for this achievement.

Sanger's reagent. The key tool is 1-fluoro-2,4-dinitrobenzene (FDNB, also called DNFB). Under mildly alkaline conditions it reacts with the unprotonated α-amino group at the N-terminus of a polypeptide, and with the ε-amino group of lysine side chains, forming a yellow 2,4-dinitrophenyl (DNP) derivative. The DNP link survives acid hydrolysis, while peptide bonds do not.

Steps of the method

1. Separating the chains: disulphide bonds holding the chains together are broken, for example by performic acid oxidation, and the chains are separated.
2. Labelling the N-terminus: each chain is treated with FDNB so that its N-terminal residue becomes a DNP-amino acid.
3. Complete hydrolysis: the labelled chain is hydrolysed with 6 M HCl at about 110 °C for around 24 hours, breaking every peptide bond.
4. Identifying the end residue: the yellow DNP-amino acid is extracted and identified by chromatography against known standards; this is the N-terminal residue.
5. Partial hydrolysis: fresh samples of the chain are cut into short overlapping fragments with dilute acid or enzymes such as trypsin, chymotrypsin and pepsin.
6. Fragment analysis: the fragments are separated by paper chromatography and electrophoresis, and the N-terminal residue and composition of each fragment are found.
7. Overlapping: the fragment sequences are lined up like pieces of a jigsaw to rebuild the full chain, and the positions of disulphide bridges are located separately.

Limitations. Acid hydrolysis destroys the rest of the chain, so only one residue is identified per labelled sample and the process cannot be repeated step by step on the same molecule. The method is slow, needs fairly large amounts of pure protein, and lysine side chains also pick up the DNP label, which must be allowed for.

Significance. Sanger's work proved that every protein has a unique, genetically fixed sequence. His overlap strategy is still used, while stepwise Edman degradation and mass spectrometry have replaced FDNB for routine sequencing.

Gel filtration chromatography and the functional diversity of proteins

Question 8 (a): Describe the principle and application of gel filtration chromatography. (7)

Question 8 (b): Explain the functional diversity of proteins. (7)

(a) Principle and application of gel filtration chromatography

Gel filtration chromatography, also called size-exclusion or molecular sieve chromatography, separates molecules according to their size and shape as they pass through a column packed with porous gel beads. Unlike ion-exchange or affinity chromatography, the molecules do not bind to the stationary phase.

Principle. The column is packed with beads of a cross-linked, hydrated gel such as Sephadex (dextran), Bio-Gel P (polyacrylamide) or Sepharose (agarose). Each bead contains pores of a controlled size. The liquid between the beads is the void volume (V₀), and the liquid inside the pores is the internal volume.

Molecules too large to enter the pores stay in the void volume, move fastest and elute first. Small molecules diffuse into the pores, follow a longer path and elute last. Molecules of intermediate size enter some pores and elute in between, so proteins leave the column in order of decreasing size.

Elution is described by the elution volume (V_e) and the partition coefficient K_av = (V_e − V₀) ÷ (V_t − V₀), where V_t is the total bed volume. Within a gel's fractionation range, V_e falls in a straight line as the logarithm of molecular mass rises, so a calibration curve of standard proteins gives the mass of an unknown.

Procedure in brief. The gel is swollen in buffer, packed without air bubbles and equilibrated. A small sample volume is loaded and eluted with the same buffer, and fractions are collected and monitored by absorbance at 280 nm.

Diagram to draw: a column of porous beads with a large molecule passing around the beads and a small molecule entering the pores, plus an elution profile showing the large-molecule peak before the small-molecule peak.

Applications

1. Purification: a standard step for separating a target protein from larger or smaller contaminants under gentle, non-denaturing conditions.
2. Native molecular mass: because the protein is not denatured, it gives the mass of the whole oligomer, which can be compared with SDS-PAGE subunit mass to reveal quaternary structure.
3. Desalting and buffer exchange: removing ammonium sulphate after salting out, or moving a protein into a new buffer, faster than dialysis.
4. Separating aggregates from monomers in quality checks of antibodies and therapeutic proteins.
5. Binding studies: separating a protein-ligand complex from unbound ligand.
6. Clean-up after labelling: removing unreacted dyes, tags or cross-linkers after chemical modification.

Its mild conditions and high recovery make gel filtration a common final polishing step in protein purification.

(b) Functional diversity of proteins

Proteins are the most functionally diverse molecules in living cells. Built from just 20 standard amino acids, each protein folds into a unique three-dimensional shape set by its sequence, and that shape decides what it can bind and do. The main functional classes are given below.

1. Enzymes: speed up almost every reaction in the cell; trypsin digests proteins and DNA polymerase copies DNA.
2. Transport proteins: carry molecules in the body or across membranes; haemoglobin carries oxygen, serum albumin carries fatty acids and GLUT transporters move glucose into cells.
3. Storage proteins: hold reserves of nutrients or ions; ferritin stores iron, casein stores amino acids in milk and ovalbumin does so in egg white.
4. Structural proteins: give strength and support; collagen in tendons and bone, keratin in hair and nails, elastin in blood vessel walls.
5. Contractile and motor proteins: produce movement; actin and myosin slide past each other in muscle, while kinesin and dynein carry cargo along microtubules.
6. Defence proteins: protect the body; antibodies recognise foreign antigens, and fibrinogen and thrombin form blood clots.
7. Regulatory proteins and hormones: control metabolism and growth; insulin lowers blood glucose and transcription factors switch genes on or off.
8. Receptor and signalling proteins: receive and relay signals; the insulin receptor and G-protein coupled receptors start signalling cascades inside the cell.
9. Toxins: some proteins, such as snake venom components and bacterial toxins, harm other organisms.

This diversity comes from three features: the enormous number of possible sequences, the ability of side chains to form precise binding sites, and the flexibility that lets many proteins change shape when they bind a ligand, as haemoglobin does during cooperative oxygen binding.

In short, almost every biological process, from digestion and movement to immunity and cell signalling, depends on proteins. That is why this course gives its whole final block to the diversity of protein function.