A. A peptide with 12 amino acids has the following amino acid composition: 2 Met, 1 Tyr, 1 Trp , 2 Glu, 1 Lys, 1 Arg, 1 Thr, 1 Asn , 1 He, 1 Cys Reaction of the intact peptide with fluorodinitrobenzene followed by acid hydrolysis creates a derivative of Ile. A specific cleavage of the intact peptide produces fragments with the following sequences: Glu-Cys-Asn-Met-Lys Met-Glu-Thr-Arg-Trp Ile-Tyr (Questions 1-5 1. Which reagent was used for the specific cleavage? a) chymotrypsin b) trypsin c) V8 protease d) cyanogen bromide 2. Which amino acids would be released when the intact peptide was treated first with V8 protease followed by treatment with cyanogen bromide? a) Glu and Met b) Glu and Lys c) Met and Lys d) Glu, Met, and Lys 3. Which treatment would result in the release of Lys and Arg from the intact peptide? a) trypsin b) trypsin followed by dansyl chloride c) trypsin followed by carboxi peptidase d) trypsin followed by mild acid 4. If this intact peptide is sequenced using the Edman degradation, which step will be part of the procedure? a) The Edman reagent will react with all 12 amino acids simultaneously. b) Lithium borohydride will react with an a-carboxyl group. c) Phenylisothiocyanate will react with an a-amino group. d) Strong acid will be used to cleave off one modified amino acid. __ If this peptide is normally part of a multimeric protein composed of four identical subunits, what procedure might be needed prior to performing the Edman degradation? a) The four subunits should be separated and sequenced individually. b) Two specific cleavages should be done to create two sets of fragments. c) Peptide bonds should be broken using hydrazine. Disulfide bonds should be reduced with mercaptoethanol.

Question

A. A peptide with 12 amino acids has the following amino acid composition: 2 Met, 1 Tyr, 1 Trp , 2 Glu, 1 Lys, 1 Arg, 1 Thr, 1 Asn , 1 He, 1 Cys Reaction of the intact peptide with fluorodinitrobenzene followed by acid hydrolysis creates a derivative of Ile. A specific cleavage of the intact peptide produces fragments with the following sequences: Glu-Cys-Asn-Met-Lys Met-Glu-Thr-Arg-Trp Ile-Tyr (Questions 1-5 1. Which reagent was used for the specific cleavage? a) chymotrypsin b) trypsin c) V8 protease d) cyanogen bromide 2. Which amino acids would be released when the intact peptide was treated first with V8 protease followed by treatment with cyanogen bromide? a) Glu and Met b) Glu and Lys c) Met and Lys d) Glu, Met, and Lys 3. Which treatment would result in the release of Lys and Arg from the intact peptide? a) trypsin b) trypsin followed by dansyl chloride c) trypsin followed by carboxi peptidase d) trypsin followed by mild acid 4. If this intact peptide is sequenced using the Edman degradation, which step will be part of the procedure? a) The Edman reagent will react with all 12 amino acids simultaneously. b) Lithium borohydride will react with an a-carboxyl group. c) Phenylisothiocyanate will react with an a-amino group. d) Strong acid will be used to cleave off one modified amino acid. __ If this peptide is normally part of a multimeric protein composed of four identical subunits, what procedure might be needed prior to performing the Edman degradation? a) The four subunits should be separated and sequenced individually. b) Two specific cleavages should be done to create two sets of fragments. c) Peptide bonds should be broken using hydrazine. Disulfide bonds should be reduced with mercaptoethanol.

Answer 1

1. The specific cleavage pattern of the peptide fragments indicates that the reagent used for the specific cleavage is chymotrypsin. Chymotrypsin cleaves peptide bonds at the carboxyl side of Tyr, Trp, and Phe.2. When the intact peptide is treated first with V8 protease followed by treatment with cyanogen bromide, the amino acids released would be Glu and Lys. V8 protease cleaves at the carboxyl side of Glu, and cyanogen bromide cleaves at the carboxyl side of Met.3. To release Lys and Arg from the intact peptide, trypsin followed by dansyl chloride would be used. Trypsin cleaves at the carboxyl side of Lys and Arg, and dansyl chloride reacts with the N-terminal amino group.4. If this intact peptide is sequenced using the Edman degradation, the step that will be part of the procedure is that strong acid will be used to cleave off one modified amino acid. In the Edman degradation method, the N-terminal amino acid is labeled with the Edman reagent, and then a strong acid is used to cleave off the modified amino acid, allowing for its identification.5. If this peptide is normally part of a multimeric protein composed of four identical subunits, the procedure that might be needed prior to performing the Edman degradation is that disulfide bonds should be reduced with mercaptoethanol. This is because disulfide bonds can interfere with the sequencing process, and reducing them with mercaptoethanol will break the disulfide bonds and allow for the sequencing of individual subunits.

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