15. A protein is purified from a cell extract. SDS-polyacrylamide gel electrophoresis is
performed for analysis of the protein using treatment with dithiothreitol (DTT) and
protease. The image of the gel is shown below. What can be inferred from the
treatment of protein with DTT and protease. Note: the red band in two lanes is the
protease used.
a. The protein has 3 protease cleavable sites
b. The protein has two polypeptides linked by a disulfide bond and two protease
sites
c. The protein has multiple protease sites
d. The protein is a single polypeptide with a disulfide bond and 3 protease sites
Correct answer: B) The protein has two polypeptides linked by a disulfide bond and two protease sites.
From the gel image:
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Ctrl (no treatment) shows a single band, indicating one apparent species under non‑reducing conditions.
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+DTT shows two bands, meaning the native protein consists of two different polypeptide chains held together by a disulfide bond; DTT reduces this bond and separates them.
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+protease (no DTT) shows multiple smaller bands derived from the intact disulfide‑linked dimer, indicating protease cleavage within the chains.
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+DTT, protease shows several low‑molecular‑weight bands but essentially two major fragment sizes, consistent with two chains, each with one effective protease cleavage site (two protease sites total). This pattern fits option B and excludes the others.
Understanding the Gel Pattern
In SDS‑PAGE, SDS denatures proteins and confers uniform negative charge, so migration mainly depends on size. DTT reduces disulfide bonds, converting disulfide‑linked multimers into separate polypeptide chains, which then appear as additional bands at different molecular weights. Proteases hydrolyze peptide bonds at specific sites, generating fragments that run as smaller bands on the gel.
On this gel, the shift from one band in Ctrl to two bands in +DTT proves that the native protein is a heterodimer (two non‑identical chains) joined by at least one disulfide bond. The protease lanes show that each chain can be cleaved, but the overall pattern suggests one principal cleavage site per chain rather than many evenly spaced sites.
Why Option B Is Correct
Option B statement
“The protein has two polypeptides linked by a disulfide bond and two protease sites.”
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Two bands in the +DTT lane show that the original single band splits into two distinct polypeptides when the disulfide is reduced. This is the hallmark of a disulfide‑linked multimer on SDS‑PAGE.
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The presence of a limited number of additional bands after protease treatment (with and without DTT) indicates a small number of specific protease cleavage sites. The pattern is consistent with one cleavage site per chain, giving two effective protease sites in the heterodimer.
Thus, the combined information from reduction and digestion supports:
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Two different polypeptide chains
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Linked by disulfide bond(s)
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Each chain offering one main protease cleavage site
Why the Other Options Are Wrong
Option A: “The protein has 3 protease cleavable sites”
If three independent protease sites were present on a single species, extensive digestion would generate more than the limited set of major bands observed, often with a ladder‑like pattern of fragments. The gel instead indicates a structured, limited fragmentation pattern connected to two distinct chains rather than three independent sites on one chain.
Also, option A does not explain the appearance of two bands only after DTT treatment, which clearly signals two polypeptides connected by disulfide bonds—something option A completely ignores.
Option C: “The protein has multiple protease sites”
“Multiple protease sites” is too vague and misses the key structural clue. The decisive observation is the DTT‑dependent splitting of the band, which tells that the protein is multimeric with disulfide linkage. While the protease lanes certainly show cleavage, the question demands a more specific structural inference, and option C does not incorporate the disulfide‑linked heterodimer revealed by the DTT lane.
Option D: “The protein is a single polypeptide with a disulfide bond and 3 protease sites”
A single polypeptide can contain intrachain disulfide bonds, but DTT reduction of such a protein does not change the band count; it only alters conformation and sometimes mobility slightly. The appearance of two separate bands with DTT means there are two distinct chains, not one chain with internal disulfides.
Furthermore, three protease sites on one chain would again yield a different fragmentation pattern than that shown, making this option inconsistent with the gel.
SEO‑Friendly Introduction
SDS‑PAGE DTT protease analysis of purified protein is a high‑yield concept for CSIR NET Life Sciences, especially in questions on protein structure and domain mapping. In this problem, a protein purified from a cell extract is analyzed on SDS‑PAGE after treatment with dithiothreitol (DTT) and a protease, and the resulting band pattern reveals whether the protein is single‑chain or multimeric and how many protease sites it contains. By reading the control, +DTT, +protease, and +DTT +protease lanes together, it becomes clear that the correct interpretation is a disulfide‑linked heterodimer with two protease cleavage sites, matching option B.
