Basically, several steps of protein purification are performed and certain obser

Question

Basically, several steps of protein purification are performed and certain observations come with these steps (A + B). Question is what these observations tell us about the protein. It’s early on in purification so doesn’t have to be conclusive, just some possible characteristics. 1. You discover a small protein containing only 10 amino acids, called interleukin-87 (IL-87), that is involved in modulating the immune response. You are interested in purifying this small peptide so that you can study its function further. The following analyses (A-E) were performed on the purified peptide to determine its sequence an structure. A. Upon reaction with fluro2.4:dinitropbenol (FDNB or Sanger DNP-Arg and DNP-Asp, leading you to believe your protein may not be pure after al. B. You then pe expected, at pH 6.25. IL87 is then treated with sithiothreitol (DTT) and the protein rform isoelectric focusing on IL-87 and observe only one band, as is again subject to isoelectric focusing. Much to your relief, you obtain two bands Explain what you know so far about the protein (after steps A and B) and why you are relieved.

Explanation / Answer

Sanger's reagent or FDNB reacts in alkaline solution with the free amino group of the N terminal amino acid residue of the polypeptide chain to form a yellow dinitrophenyl derivative (DNP)

FDNB + polypeptide chain --------- yellow colored DNP derivative + HF

In the question we obtain two DNP derivative DNP Arg and DNP Asp. It is clear that the protein is not purified as there are a mixture of proteins since their N terminal is producing DNP derivative after reaction with FDNB or it has more than one polypeptide chain.

Isoelectric focusing restricts the movement of proteins at their isoelectric points, the pH at which the protein has no charge or is electrically neutral.  

Since Arg is positively charged amino acid and Asp a negatively charged amino acid, upon isoelectric focusing the two DNP derivatives should show two different bands with different pIs(isoelectric points). The pI of arginine is around 10 and that of Asp is 2.8 one being basic and other acidic.

However according to the question the observed band was just one near 6.25. This concludes that the sample has a protein with two polypeptide chain

Further DTT treatment lead to the visualization of our two expected bands of Arg lead polypeptide and Asp lead polypeptide

DTT is known as a protective agent which prevents intra molecular and intermolecular interaction between cysteine group of proteins. It also reduces the disulfide bonds of proteins.

So until now the only thing that could be concluded is the presence of two polypeptide chains of protein with N terminal as Asp and Arg

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