Q.51
The following polypeptide chain was sequentially treated with dithiothreitol, cyanogen bromide, and trypsin.
Phe-Trp-Lys-Tyr-Met-Gly-Ala-Cys-Cys-Pro-Met-Asp-Gly-Arg-Phe-Ala-Gly-Trp
The total number of fragments expected at the end of complete digestion of the polypeptide are _____ .
(consider that none of the reagents interfere with each other’s activities)
The polypeptide chain produces 7 fragments after sequential treatment with dithiothreitol (DTT), cyanogen bromide (CNBr), and trypsin.
DTT first reduces the disulfide bond between the two adjacent Cys residues, separating the chain into two linear polypeptides without creating new fragments. CNBr then cleaves specifically at the C-terminal side of each methionine (Met), generating distinct pieces from both chains. Trypsin subsequently cuts at the C-terminal side of lysine (Lys) and arginine (Arg) residues in those CNBr fragments.
Step-by-Step Digestion Process
Dithiothreitol (DTT) Treatment
DTT reduces the Cys-Cys disulfide bond between positions 8-9, yielding two separate chains:
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Chain 1: Phe-Trp-Lys-Tyr-Met-Gly-Ala-Cys (1-8)
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Chain 2: Cys-Pro-Met-Asp-Gly-Arg-Phe-Ala-Gly-Trp (9-19)
Cyanogen Bromide (CNBr) Cleavage
CNBr cleaves after Met residues (positions 5 and 12):
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From Chain 1: Phe-Trp-Lys-Tyr-Met | Gly-Ala-Cys (2 fragments)
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From Chain 2: Cys-Pro-Met | Asp-Gly-Arg-Phe-Ala-Gly-Trp (2 fragments)
Total: 4 CNBr fragments. No interference occurs as reduced Cys residues do not affect CNBr specificity.
Trypsin Digestion
Trypsin cleaves after Lys (position 3) and Arg (position 16):
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Phe-Trp-Lys | Tyr-Met (from first CNBr fragment) → 2 fragments
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Gly-Ala-Cys → 1 fragment (no Lys/Arg)
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Cys-Pro-Met → 1 fragment (no Lys/Arg)
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Asp-Gly-Arg | Phe-Ala-Gly-Trp → 2 fragments
Total: 7 final fragments. Trypsin does not cleave Lys-Pro bonds, but no such sites exist here.
Fragments Summary Table
| Original Position | CNBr Fragment | Trypsin Fragments |
|---|---|---|
| 1-5 | Phe-Trp-Lys-Tyr-Met | Phe-Trp-Lys Tyr-Met |
| 6-8 | Gly-Ala-Cys | Gly-Ala-Cys |
| 9-12 | Cys-Pro-Met | Cys-Pro-Met |
| 13-19 | Asp-Gly-Arg-Phe-Ala-Gly-Trp | Asp-Gly-Arg Phe-Ala-Gly-Trp |
Introduction to Polypeptide Sequential Digestion
Sequential enzymatic and chemical digestion determines polypeptide fragments in protein sequencing, vital for CSIR NET Life Sciences. This method uses DTT for disulfide reduction, CNBr for methionine-specific cleavage, and trypsin for Lys/Arg cuts, yielding precise polypeptide fragments without reagent interference.
Key Reagents in Protein Fragmentation
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Dithiothreitol (DTT): Breaks Cys-Cys bonds, linearizing chains.
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Cyanogen Bromide (CNBr): Cleaves C-terminal to Met, forming homoserine lactone.
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Trypsin: Hydrolyzes after Lys/Arg (except before Pro), generating tryptic peptides.
Application in CSIR NET Exams
This polypeptide sequential digestion question tests cleavage site identification. Reduced Cys allows independent chain processing, leading to 7 polypeptide fragments. Practice similar problems for competitive exams.
