Q.No. 52 Protein A and protein B form a covalent complex.
Gel filtration chromatography of this complex showed a peak corresponding to 200 kDa.
SDS-PAGE analysis of this complex, with and without beta-mercaptoethanol,
showed a single band corresponding to molecular weight 50 and 25 kDa, respectively.
Given that the molecular weight of protein A is 25 kDa,
the molecular weight of protein B is ____________ kDa.
Protein A (25 kDa) and protein B form a covalent complex that appears as 200 kDa in gel filtration chromatography, indicating the native molecular weight of the intact complex. SDS-PAGE without β-mercaptoethanol shows a 50 kDa band, revealing a disulfide-linked dimer, while with β-mercaptoethanol it shows 25 kDa, corresponding to the monomer subunit. Thus, protein B must also be 25 kDa, forming a homodimer complex of four 50 kDa units (2A:2B) totaling 200 kDa.
Technique Principles
Gel filtration chromatography separates native proteins by size, so the 200 kDa peak reflects the intact complex’s hydrodynamic volume. SDS-PAGE denatures proteins; without reducing agent (β-mercaptoethanol), disulfide bonds persist, yielding the 50 kDa band as a covalently linked A-B heterodimer. Adding β-mercaptoethanol breaks these bonds, separating subunits to 25 kDa for protein A (and B).
Step-by-Step Solution
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Native complex MW = 200 kDa (gel filtration).
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SDS-PAGE (+β-ME): Single 25 kDa band means both A and B subunits are 25 kDa.
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SDS-PAGE (-β-ME): Single 50 kDa band means each linked pair (A-B) totals 50 kDa via disulfide.
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Complex composition: Four A-B heterodimers (4 × 50 kDa = 200 kDa).
Protein B = 25 kDa.
Introduction: Protein A and Protein B Covalent Complex Analysis
In protein A and protein B covalent complex studies, gel filtration chromatography reveals 200 kDa native size while SDS-PAGE shows 50 kDa without β-mercaptoethanol and 25 kDa with it. This IIT JAM-level question tests biochemistry techniques for molecular weight determination. Protein A is given as 25 kDa; find protein B.
Key Concepts Explained
Gel filtration measures native protein complexes by size exclusion. SDS-PAGE with reducing agents like β-mercaptoethanol breaks disulfide bonds in covalent complexes.
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Native State (Gel Filtration): 200 kDa intact complex elutes as one peak.
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Non-Reduced SDS-PAGE: Disulfide-linked A-B dimer at 50 kDa (25+25).
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Reduced SDS-PAGE: Monomers at 25 kDa confirm identical subunits.
Detailed Calculation
| State | MW Observed | Interpretation |
|---|---|---|
| Gel Filtration | 200 kDa | Tetramer of dimers (4×50) |
| SDS-PAGE (-β-ME) | 50 kDa | A-B heterodimer |
| SDS-PAGE (+β-ME) | 25 kDa | A or B monomer |
Since Protein A = 25 kDa and reduced band is single 25 kDa, Protein B = 25 kDa. Complex: (A-B)₂ tetramer.
Exam Tips
Practice interpreting gel filtration vs SDS-PAGE for covalent vs non-covalent complexes. Single bands indicate uniform subunits. Ideal for IIT JAM Biotechnology preparation.
