1. You discover a small protein containing only 10 amino acids, called interleuk
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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 and structure. A. Upon reaction with fluro-2.4:dinitrophenol (FDNB or Sanger reagent), you obtain DNP-Arg and DNP-Asp, leading you to believe your protein may not be pure after all. B. You then perform isoelectric focusing on IL-87 and observe only one band, as expected, at pH 6.25. IL-87 is then treated with dithiothreitol (DTT) and the protein is again subject to isoelectric focusing. Much to your relief, you obtain two bands, one at pH of 10 and one at pH of 2.85 Explain what you know so far about the protein (after steps A and B) and why you are relieved.Explanation / Answer
In sanger sequencing using fluoro-2,4-dinitrophenol only the N-terminal amino acid can be determined ie the first amino acid. The sangers degradation method involve a reaction between fluoro-2,4-dinitrophenol and N-terminal amino acid to form a yellow-coloured derivative of sanger's reagent and N terminal amino acid. Acid or enzymatic hydrolysis of each and every peptide bond is done. This results in a mixture of unlabelled amino acids and DNP-labelled N-terminal amino acid. In A two DNP derivative are seen, one is DNP-Arg and other is DNP-Asp. Therefore our peptide IL-87 may contain either arginine or aspartate at its N-terminus.
B. As seen in A our protein is contaminated with other protein. In B when IL-87 is treated with DTT two bands are seen. DTT is a reducing agent which reduces disulfide bonds connecting two proteins and denatures protein into individual subunits. One band is at ph10 and other is at ph 2.85. Without treatment with DTT only one band is seen at 6.25. pI is the pH at which proteins stop migrating as net charge in zero.
After considering both A and B observations it can be concluded that IL-87 is a heterodimer as two bands of different pI are seen in Isoelectric focussing gels after treatment with DTT . The pI of IL-87 should be 6.25 as a single band is seen at pH 6.25 before DTT treatment.
In A two DNP-derivatives were seen: DNP-arg and DNP-asp. One of the subunit may be having arginine at N-terminal and other might be having aspartate at its N-terminal.
We are relieved because our protein IL-87 is not contaminated, instead it is a heterodimer. That is why it gave two DNP derivative and two bands of different pI value when treated with DTT.
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