10. An enzyme examined by means of gel filtration in aqueous buffer at pH 7.0 had an apparent molecular weight of 160,000.
When examined by gel electrophoresis in SDS solution, a single band of apparent molecular weight 40,000 was formed.
Explain these findings.
A. Enzyme comprises single units of Mr = 160,000
B. Enzyme comprises two identical subunits of Mr = 40,000
C. Enzyme comprises four identical subunits of Mr = 40,000
D. Enzyme comprises eight identical subunits of Mr = 20,000
Gel filtration at pH 7 measures native molecular weight (160 kDa), while SDS-PAGE denatures proteins into individual polypeptide chains (40 kDa single band), revealing quaternary structure.
Technique Comparison
Gel Filtration (Native, pH 7): Measures hydrodynamic size of intact oligomer. 160 kDa = native quaternary structure.
SDS-PAGE: SDS denatures + coats proteins with uniform negative charge; β-mercaptoethanol breaks disulfide bonds. Single 40 kDa band = all subunits identical, 40 kDa each.
Calculation
160 kDa ÷ 40 kDa = 4 subunits
Option Analysis
A. Enzyme comprises single units of Mr = 160,000
Wrong. Single 160 kDa polypeptide would show 160 kDa band in SDS-PAGE, not 40 kDa.
B. Enzyme comprises two identical subunits of Mr = 40,000
Incorrect. 2 × 40 kDa = 80 kDa native, not 160 kDa observed.
C. Enzyme comprises four identical subunits of Mr = 40,000
Correct. 4 × 40 kDa = 160 kDa native. Single SDS band confirms identical subunits.
D. Enzyme comprises eight identical subunits of Mr = 20,000
Wrong. SDS-PAGE shows 40 kDa bands, not 20 kDa.
Correct Answer
C. Enzyme comprises four identical subunits of Mr = 40,000
Protein Analysis Table
| Technique | Conditions | Measures | Result | Interpretation |
|---|---|---|---|---|
| Gel Filtration | Native, pH 7 | Quaternary structure | 160 kDa | Native tetramer |
| SDS-PAGE | Denaturing | Polypeptide MW | 40 kDa (single band) | Identical 40 kDa subunits |
GATE Prep Essential
Classic subunit stoichiometry question pattern:
Native MW ÷ SDS MW = # identical subunits
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Single SDS band = identical subunits
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Multiple SDS bands = non-identical subunits
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Same MW in both = monomer
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No β-mercaptoethanol info = assume disulfides broken (standard SDS-PAGE)
Examples to memorize:
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Hemoglobin: 64 kDa native ÷ 16 kDa SDS = 4 subunits (2α2β)
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LDH: 140 kDa native ÷ 35 kDa SDS = 4 subunits
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IgG: 150 kDa native → 50 kDa heavy + 25 kDa light (4 chains total)
Pro Tip: pH 7 buffer + “aqueous” confirms native conditions. SDS always denatures to polypeptide level. Perfect 4x multiple = tetramer (most common enzyme oligomeric state).
