The electrophoretic separation of proteins
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The electrophoretic separation of proteins
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[ID] => 560274
[post_author] => 12815
[post_date] => 2025-01-14 06:10:40
[post_date_gmt] => 2025-01-14 11:10:40
[post_content] => Practice Passage (Question 1-5)
*This passage is the property of Khan Academy and has been reformatted into an AAMC-style interface in their entirety by MedLife Mastery. MedLife Mastery does not endorse and is not an affiliate of Khan Academy.
Adrenocorticotropic hormone (ACTH) is a 39 amino acid polypeptide secreted by the anterior pituitary gland. ACTH is derived from the 241 amino acid polypeptide pro-opiomelanocortin (POMC) via the activity of endoproteases, which catalyze the hydrolysis of peptide bonds between adjacent basic residues of polypeptide chains (see figure one). In addition to ACTH, several other polypeptides are derived from POMC. Table one lists some of these breakdown products. Of particular interest is alpha-MSH, a melanocyte-stimulating hormone consisting of the first 13 amino acid residues of ACTH. Addison’s disease (primary adrenal insufficiency), resulting from the under-production of cortisol by the adrenal cortex, is associated with increased serum levels of ACTH and increased secretion of the pigment melanin by melanocytes.
Table one. Breakdown products of pro-opiomelanocortin (POMC). "Position" is measured from the N-terminus amino acid residue of POMC to the C-terminus residue. "Length" refers to the number of amino acid residues in the protein of interest.
Figure one. Simplified diagram of change in free energy during endoprotease catalyzed hydrolysis of POMC.
The development of reliable assays to assess circulating levels of hormones like ACTH is of importance in the clinic. Perhaps the most straightforward way to assess the presence or absence of a particular protein in a given sample is to use SDS-page, which spatially separates proteins in a sample according to molecular weight. In SDS-page, a given sample is heated in a solution containing sodium dodecyl sulfate (SDS) and betamercaptoethanol (BME). SDS is a negatively charged surfactant that binds polypeptides in a quantity directly proportional to the length of the polypeptide. BME is a strong reducing agent that is able to disrupt aspects of the tertiary and quaternary structure of proteins. Once treated with SDS, BME, and heat, the protein sample is loaded into a polyacrylamide gel contained in a pH buffered solution (note: polyacrylamide gels consist of a chemical matrix with regularly shaped pores through which molecules can migrate). A constant voltage is applied across the gel, which causes the movement of proteins through the gel. Assuming a constant voltage, the rate of migration of a protein in SDS-page is inversely proportional to the logarithm of its molecular weight.
[post_title] => The electrophoretic separation of proteins
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[question] => Why do patients suffering from Addison’s disease display a phenotype associated with hypersecretion of melanin by melanocytes without increased plasma levels of α-MSH, the primary melanocyte stimulating hormone in humans?
[value] => Array
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[answer] => 2
[description] => Reason for the Correct Answer:
The passage states the Addison’s disease is associated with decreased levels of glucocorticoids.
The passage does not state that Addison’s disease is in any way related to a genetic mutation leading to constitutive activity of melanocytes.
The sequence of amino acids in a protein determines the function of the protein, thus two proteins that have similar or identical sequences of amino acids may be able to function in similar ways by binding similar receptors.
The similarity in the amino acid sequence of ACTH and α-MSH allows ACTH to bind α-MSH receptors under certain conditions.
)
[answers] => Array
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[0] => Array
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[each_answer] => A.Addison’s disease is caused by a genetic mutation that renders melanocytes constitutively active.
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[1] => Array
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[each_answer] => B.The similarity in the amino acid sequence of ACTH and α-MSH allows ACTH to bind α-MSH receptors under certain conditions.
)
[2] => Array
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[each_answer] => C.Addison’s disease is associated with increased levels of glucocorticoids, which can be interconverted to α-MSH by prohormoneconvertases secreted by melanocytes.
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[3] => Array
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[each_answer] => D.Addison’s disease is associated with decreased levels of glucocorticoids, which stimulate the production of ACTH via a positive feedback loop.
)
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[1] => Array
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[quiz_unique_key] => 3873426850
[question] => Why does nucleic acid electrophoresis not require the use of SDS?
[value] => Array
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[answer] => 4
[description] => Reason for the Correct Answer:
SDS is used to ensure that polypeptides have a negative charge proportional to the length of the molecule.
Altering the pH of a solution can increase or decrease the concentration of proton donors and/or acceptors, which can effect the net charge on DNA molecules.
Phosphate groups, which participate in the phosphodiester bonds linking nucleotides in DNA, are negatively charged.
DNA molecules already have a net negative charge proportional to their length.
)
[answers] => Array
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[0] => Array
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[each_answer] => A.Under physiological conditions, DNA exists in its lowest oxidation state.
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[1] => Array
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[each_answer] => B.The pH of a solution will not alter the net charge on DNA molecules.
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[2] => Array
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[each_answer] => C.DNA molecules already have a net positive charge proportional to their length.
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[3] => Array
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[each_answer] => D.DNA molecules already have a net negative charge proportional to their length.
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[2] => Array
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[quiz_unique_key] => 83407773
[question] => Why is the reverse reaction of endoprotease catalyzed hydrolysis of POMC (the endoprotease catalyzed condensation of ACTH with the remaining polypeptide fragment of POMC) generally not observed under physiological conditions?
[value] => Array
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[answer] => 2
[description] => Reason for the Correct Answer:
Enzymes catalyze chemical reactions by lowering the activation energy required for the reaction to occur.
The energy required to drive biochemical reactions is obtained from the surrounding environment.
All chemical reactions are reversible, at least in principle.
The endoprotease in question lowers the activation energy for the condensation reaction, but not enough for it to occur under physiological conditions.
)
[answers] => Array
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[0] => Array
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[each_answer] => A.The endoprotease responsible for the hydrolysis reaction does not lower the activation energy for the reverse condensation reaction.
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[1] => Array
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[each_answer] => B.The endoprotease in question lowers the activation energy for the condensation reaction, but not enough for it to occur under physiological conditions.
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[2] => Array
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[each_answer] => C.The endoprotease responsible for the reaction undergoes a permanent alteration to its active site during the hydrolysis reaction.
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[each_answer] => D.The endoprotease responsible for the hydrolysis reaction lowers the Gibbs free energy of ACTH but not POMC.
)
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[quiz_unique_key] => 2377279144
[question] => Why is SDS-page carried out in a pH buffered solution?
[value] => Array
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[answer] => 2
[description] => Reason for the Correct Answer:
SDS-page electrophoresis requires the disruption of the native conformations of the proteins of interest.
SDS-page is not carried out under physiological conditions (BME and SDS are not present in living systems).
SDS-page uses a voltage gradient to spatially separate electrically charged proteins.
SDS-page is carried out in a pH buffered solution to ensure that the proteins being separated maintain the desired electrical charge.
)
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[0] => Array
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[each_answer] => A.Using a pH buffered solution disrupts protein-protein interactions that occur under physiological conditions, which are not pH buffered, allowing for complete separation across the gel.
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[1] => Array
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[each_answer] => B.Using a pH buffered solution ensures that SDS-bound polypeptides maintain the desired electrical charge.
)
[2] => Array
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[each_answer] => C.Using a pH buffered solution maintains the native conformations of the proteins being separated.
)
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[each_answer] => D.Using a pH buffered solution prevents the autoionization of water, inhibiting the production of reactive hydronium and hydroxide species.
)
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[4] => Array
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[quiz_unique_key] => 2261298308
[question] => If SDS-page is performed with a sample containing ACTH and α-MSH, how far will α-MSH migrate as compared to the distance ACTH migrates?
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[answer] => 3
[description] => Reason for the Correct Answer:
SDS, BME, and heat disrupt the molecular forces that confer conformational shape on polypeptides.
Distance of migration depends on molecular weight.
ACTH is heavier than α-MSH , since, according to table one, ACTH has 26 more amino acids than α-MSH .
The distance α-MSH migrates is greater than the distance ACTH migrates.
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[each_answer] => A.The distance α-MSH migrates is equal to one-third the distance ACTH migrates.
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[each_answer] => B.The distance α-MSH migrates is equal to the natural logarithm of the distance ACTH migrates.
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[each_answer] => C.The distance α-MSH migrates is greater than the distance ACTH migrates.
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[each_answer] => D.More information is needed, since the distance of migration in an SDS-page gel also depends on the conformational shape of the polypeptides.
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