Q.50 In a denaturing polyacrylamide gel electrophoresis experiment, pure intact adult
human hemoglobin will yield ________(number) bands.
Pure intact adult human hemoglobin, or HbA (α₂β₂ tetramer), separates into 2 bands on denaturing polyacrylamide gel electrophoresis due to dissociation into α and β globin subunits.
Hemoglobin Structure
Adult hemoglobin consists of two identical α-globin chains (141 amino acids, ~15.1 kDa) and two identical β-globin chains (146 amino acids, ~15.9 kDa), forming a functional tetramer. In native conditions, subunits associate non-covalently, but denaturation disrupts this. The slight mass difference allows separation under denaturing conditions.
Denaturing PAGE Mechanism
Denaturing polyacrylamide gel electrophoresis, typically SDS-PAGE, uses SDS detergent to unfold proteins into linear polypeptides coated with negative charge, plus reducing agents like DTT or β-mercaptoethanol to break disulfide bonds. Migration depends solely on molecular mass through the polyacrylamide matrix, resolving subunits by size. For hemoglobin, tetramer dissociates completely into individual α and β chains.
Expected Bands Analysis
Two distinct bands appear: one for α-chains (faster migrating, lower mass) and one for β-chains (slower). Standard protocols resolve these due to ~5% mass difference, though low-resolution gels may smear them as one. CSIR NET contexts confirm 2 bands for pure HbA, excluding minor δ-chains (~0.5-1% in adults).
| Option | Bands | Explanation | Correct? |
|---|---|---|---|
| 1 | One band | Intact tetramer (native PAGE); subunits unresolved or identical mass assumed. | No |
| 2 | Two bands | α (~15 kDa) and β (~16 kDa) subunits separated by mass. | Yes |
| 4 | Four bands | Individual subunits counted separately despite identical pairs. | No |
| >4 | Multiple | Impurities or degradation; pure intact HbA yields only 2. | No |
CSIR NET Exam Relevance
This question tests understanding of protein quaternary structure dissociation in SDS-PAGE, key for molecular biology units. Pure intact sample rules out fetal (γ) or minor variants; reducing conditions ensure full denaturation. Practice gels with molecular markers confirm ~15-16 kDa bands.
