A. Chromatofocusing

B. Gel filtration chromatography

C. Native PAGE

D. Analytical isoelectric focusing

Chromatofocusing is the ideal technique for large-scale purification of isozymes differing by a single charged amino acid.

This MCQ tests charge-based separation methods suitable for preparative scale.

Correct Answer

A. Chromatofocusing [ from prior context]

Option Analysis

Isozymes A and B differ by one positively charged amino acid (e.g., Lys vs. neutral), creating a small pI difference (~0.1-0.5 units). Large-scale purification requires charge exploitation at preparative volumes.

• A. Chromatofocusing: Correct. This ion-exchange chromatography variant creates a predefined pH gradient (pH 9→4) on anion/cation exchangers. Isozymes focus (stop migrating) at their exact pI positions and elute sharply. Perfect for small pI differences; scalable to hundreds of mL bed volumes for industrial purification. [prior -27 context]

• B. Gel filtration chromatography: Incorrect. Separates by size/shape only—charge differences are irrelevant. Both isozymes (similar MW) co-elute together. Not suitable for charge-based separation. [prior ,5]

• C. Native PAGE: Incorrect. Polyacrylamide gel electrophoresis separates by charge/mass but is analytical (small gel volumes, μg scale). Cannot handle large-scale (grams/liters) purification required here.

• D. Analytical isoelectric focusing: Incorrect. IEF separates at pI positions in pH gradients but is analytical/preparative small-scale (rotor/tube gels). Not designed for industrial large-scale purification.

Large-scale purification isozymes single positively charged amino acid is critical for enzyme production in biotech/pharma. A single Lys/Arg difference creates exploitable pI variation for chromatofocusing.

Chromatofocusing Principle

Anion/cation exchanger equilibrated at high pH (9.0). Polybuffer (ampholyte mimic) creates internal pH gradient (9→4) during elution. Proteins bind → migrate → focus at pI → elute as narrow peaks (ΔpI = 0.05 resolvable).

Scalable: 100 mL → 10 L columns routine. Recovery: >90%. Resolution: Better than standard ion-exchange for close pI. [biochem principle]

Why Other Techniques Fail Large Scale

Real-World Application

LDH isozymes (heart M4 vs. muscle H4) differ by charged residues → chromatofocusing baseline resolves. Industrial: Alkaline phosphatase isozymes for diagnostics.

GATE Exam Strategy

Pattern: “Large-scale” eliminates PAGE/IEF. “Single charged AA” eliminates size methods. Answer: Charge gradient chromatography (chromatofocusing > ion-exchange).

This PYQ tests scale + separation principle integration—key for bioprocess engineering questions.