Question

Question 3: Arg-Cys-Met-Ala-Cys-Gly-Arg-Pro-Asn-Tyr-Leu-Trp-Ala-Ile-His-Phe-Ser-Cys-Lys a. What would happen if this peptide were to be incubated with dinitrofluorobenzene (FDNB) followed by 6M HCl hydrolysis at 1100C for 24 hrs. What labeled product(s) would be detected? Consider the following pepide: What would happen if the peptide were treated with CNBr? What would the products be? Why? b. What would happen if the peptide were treated with chymotrypsin? What would the c. products be? Why? Arg-Cys-Met-Ala-Cys-Gly-Arg-Pro-Asn-Tyr, Leu-Trp, Ala-Ile-His-Phe, and Ser-Cys- Lys Chymotrypsin cleaves the peptide bond at the C terminal side of aromatic amino acids. d. What would happen if the peptide were treated with trypsin? What would the products be? Why? What would happen if the peptide were treated with DTT and iodoacetate? What would be the products and why? e.

please explain each question thoroughly. thanks

Answer 1

Answer a)

1. The method mentioned is Sanger's Method of detection of amino acid. The above peptide sequence has the amino acid Arginine at the N-terminal end where the amino group is present as free end.
2. Dinitrofluorobenzene basically attaches to the amino group present at the N-terminal end. This attachment is associated with developing a Fluorescence later during the process.
3. In the next step, it is treated with an acid ( 6M HCL ) which in turn is responsible for cleavage of the remaining peptide chain. This step is needed for hydrolysis to occur which breaks the peptide bond leading to release of water molecule and hence termed as acid hydrolysis.
4. Finally, the labelled amino acid will be Arginine which will be stated as the terminal amino acid.

Answer b:-

1. Cyanogen bromide basically cleaves the carboxy end of the amino acid Methionine and hence there would be formation of two shorter fragment from the original peptide.
2. First peptide:- Arg-Cys-Met
3. Second peptide:- Ala-Cys-Gly-Arg-Pro-Asn-Tyr-Leu-Trp-Ala-Ile-His-Phe-Ser-Cys-Lys
4. This is due to the nucleophilic substitution reaction occuring with respect to the Carbon atom of CnBr which has more affinity towards electrons.