1.An SDS-PAGE gel is run of proinsulin and insulin. Samples were treated with -m
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Question
1.An SDS-PAGE gel is run of proinsulin and insulin. Samples were treated with -mercaptoethanol (a reageant that breaks down disulfide bonds) prior to electrophoresis. What size bands should be present in proinsulin? What size bands should be present for insulin? Explain your answers.
2. A denaturation/renaturation experiment was carried out with insulin (similar to the one carried out by Anfinsen with ribonuclease). However, in contrast to Anfinsen’s results, only about 10% of the activity of insulin was recovered when urea and –mercaptoethanol were removed by dialysis. (This is the level of activity you would expect if the disulfide bridges paired randomly.) In contrast, if the experiment is repeated with proinsulin, full activity is restored upon renaturation. Explain these observations.
4.Explain why the pancreas would produce the protein proinsulin, which contains many additional amino acids that are later removed, instead of directly producing insulin.
5.Does the C chain have any biological activity on its own, apart from its role in proinsulin? Search for information and summarize your findings with an appropriate citation from a primary literature reference.
51 60 57 58 A Chain-C peptide Junction 63 LEU C-PEPTIDE LYS GLY 2 CLE VAL GLU GLN A-CHAIN CYS CYS SER CTS GERMLEU TYR GLN LEU GLU ASN ASN PHE 11 12 13 21 14 15 16 17 18 19 VAL insulin Antise ASN GLN LEU B-CHAIN CYS GLY SER HIS HE LEU GLY VAL ARG 10 GLU ALA 11 12 LEU 13 14 16 1 GL VAL 41 VAL LEU 37 ASP 36 ARG 32 31 THR LYS PRO THR TYR B Chain-C peptide JunctionExplanation / Answer
1. Samples when treated with mercaptoethanol, gets cleaved at disulfide bonds. Proinsulin has 86 aminoacids. Assuming each aminoacid has an average molecular weight of 110 Da, proinsulin containing 86 aminoacids would have molecular weight of 9460 Da and thus gives band at this size.
Whereas insulin has two chains with 30 aminoacids and 21 aminoacids. Therefore, when treated with mercaptoethanol, disulfide bonds between these two chains get cleaved and gives two bands, one at 3300 Da and the other at 2310 Da.
2. Removal of urea and mercaptoethanol restores full activity of proinsulin but only 10% of insulin activity.
This is because, proinsulin contains C-peptide that enables proper spacing and alignment of A- and B-peptides so that disulfide bonds are formed at correct positions to give full activity.
Whereas, insulin does not contain C-peptide. Hence, removal of denaturants may lead to disulfide bonds at wrong positions such as between 6-Cys of A-peptide and 7-Cys of B-peptide or 7-Cys of A-peptide and 19-cys of B-peptide. Therefore, its activity is not restored. The disulfide bond formation is random.
3. Proinsulin is an inactive form of insulin. Therefore, injecting proinsulin would not serve the purpose of owering blood glucose level.
4. Pancreas secretes proinsulin but not insulin. Proinsulin is an inactive form and has longer half life compared to insulin. Insulin needs to function only at high blood glucose levels. Therefore, proinsulin gets converted to insulin during high glucose level. Or it converts to insulin only under its favourable conditions.
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