The following information was obtained on a neuropeptide isolated from Saguinus
ID: 226782 • Letter: T
Question
The following information was obtained on a neuropeptide isolated from Saguinus midas. use the information to determine the primary sequence of the poly peptide, how many individual chains it is made up of, and if/where disulfide bonds exist.
1) Amino acid analysis revealed the following composition for the decapeptide: Arg, Cys, Cys, Cys, Lys, Lys, Met, Pro,Tyr, Trp
2) Reverse phase HPLC was run on the peptide under both reducing and non-reducing conditions and a single peak was observed in either case.
3) Treatment of the decapeptide with FDNB followed by acid hydrolysis resulted in the detection of DNB-Trp. TReatment of the Decapeptide with hydrazine resulted in free cysteine and the hydrazine-nonapeptide derivative. They only could be seperated by HPLC if the sample was reduced.
4) Treatment of the decapeptide with chymotrypsin resulted in the generation of freee tryptophan, a dipeptide, and a heptapeptide. Treatment of the decapeptide with pepsin showed no clevage products being generated.
5) treatment of the decapeptide with CNBr resulted in 2 pentapeptides. The pentapeptides were reacted with FDNB followed by acid hydrolysis and DNB-Trp and DNB-Cys were identified.
6) The pentapeptide with Cyc at its N-terminus was analyzed further. Treatment of the pentapeptide wth submaxillary protease resulted in the production of free cysteine and a tetrapeptide that could only be seperated if the sample was reduced after the clevage.
7) Additional treatment of the pentapeptide woth Cys at its N-terminus with trypsin resulted in a dipeptide and a tripeptide under non-reducing conditions. under reducing conditions, treatment with trypsin generated 2 dipeptides and free cysteine.
Explanation / Answer
1) Decapeptide composition is Arg, Cys, Cys, Cys, Lys, Lys, Met, Pro,Tyr, Trp
2) Reverse phase HPLC was run on the peptide under both reducing and non-reducing conditions and a single peak was observed in either case.
So, there are no disulphide bonds. If there are disulphide bonds, retention time/peak size will be different between HPLC in reducing and non-reducing conditions.
3) Treatment of the decapeptide with FDNB followed by acid hydrolysis resulted in the detection of DNB-Trp. This indicates there is one peptide and the amino-terminus aminoacid is tryptophan. FDNB reacts with N-terminus aminoacid only.
4) Treatment of the Decapeptide with hydrazine resulted in free cysteine and the hydrazine-nonapeptide derivative. They only could be separated by HPLC if the sample was reduced.
Hydrazine treatment releases c terminal amino acid intact. In this case, c terminal amino acid is cysteine.
So, based on observation 3 and 4, peptide sequence is
Trp-x-x-x-x-x-x-x-x-Cys
5) Treatment of the decapeptide with chymotrypsin resulted in the generation of free tryptophan, a dipeptide, and a heptapeptide. Treatment of the decapeptide with pepsin showed no clevage products being generated.
Chymotrypsin cleaves Carboxyl Side of Aryl Amino Acids (e.g. Phe, Tyr & Trp). So, the peptide in question has Tyr, Trp. We can expect that chymotrypsin cleaves carboxyl side of Tyr as well as Trp. We already know that n terminal amino acid is Trp. We got it as a free amino acid. In addition, we got a dipeptide and a heptapeptide. It means, position of Tyr is either 3 or 8
So, the expected sequence is
Trp-x-(x/Tyr)-x-x-x-x- (x/Tyr) -x-Cys
Pepsin cleaves at the amino end of Phe, Tyr, Trp if they are not preceded by proline. We already know that N terminal amino acid of the decapeptide is Trp. So, there is nothing to cleave at its amino side. The next target position is amino side of Tyr. But, pepsin did not cleave the peptide. It means, proline is present in the amino side of Tyr.
So, the expected sequence is
Trp-x-(x-x/Pro-Tyr)-x-x- (x-x/Pro-Tyr) -Cys
6) treatment of the decapeptide with CNBr resulted in 2 pentapeptides. The pentapeptides were reacted with FDNB followed by acid hydrolysis and DNB-Trp and DNB-Cys were identified.
Cyanogen bromide (CNBr) specifically cleaves Met residues at the C-end. So, the amino acid in fifth position is Met.
As mentioned in point 3, FDNB reacts with N-terminus aminoacid only. After treating both pentapeptides, we got DNB-Trp and DNB-Cys. We know that N terminal amino acid of the decapeptide is Trp. So, the amino acid in sixth position is Cys.
So, the expected sequence is
Trp-x-(x-x/Pro-Tyr)-Met-Cys-x- (x-x/Pro-Tyr) -Cys
6) The pentapeptide with Cyc at its N-terminus was analyzed further. Treatment of the pentapeptide wth submaxillary protease resulted in the production of free cysteine and a tetrapeptide that could only be seperated if the sample was reduced after the clevage.
Submaxillary protease cleaves c terminal to Arg residues. We already know that N terminal amino acid of this penta-peptide is Cys. So, the residue next to thes Cys is Arg.
So, the expected sequence is
Trp-x-(x-x/Pro-Tyr)-Met-Cys-Arg- (x-x/Pro-Tyr) -Cys
7) Additional treatment of the pentapeptide with Cys at its N-terminus with trypsin resulted in a dipeptide and a tripeptide under non-reducing conditions. under reducing conditions, treatment with trypsin generated 2 dipeptides and free cysteine.
So, we are talking here at this part of decapeptide.
Cys-Arg- (x-x/Pro-Tyr) -Cys
Trypsin cleaves at the C-end of positively charged amino acids (Arg and Lys) if the next residue is not a proline.
Under reducing conditions, treatment with trypsin generated 2 dipeptides and free cysteine. So, Cys-Arg forms one dipeptide. Free Cys is probably C terminal Cys. Then, Pro-Tyr pair is present in other hals of the deca peptide. So, the ninth position is Lys.
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