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d) Cleavage by trypsin followed by gel-filtration chromatography gives the expected 6 products, which you sequence (shown in order of emergence from column): T-1 ETMESSAGEFGR T-2 SQTWALDHSECR T-4 TCR S-5 TME Think About It: T-5 NP e) Cleavage by Staph. aureus V8 protease followed by gel-filtration chromatography gives the expected 7 products, which you sequence (shown in order of emergence from column): S-1 RSQTWALD S-2 FGRGPQD S-3 NKTCRNP S-6 CRE T-3 GPQDNK S-7 HSE T-6 R S-4 SSAGE 1. Deduce the primary structure of this polypeptide. 2. Why would cyanogen bromide not be a good choice as a cleavage reagent? 3. Can you account for the order of elution of trypsin digest peptides from gel-filtration chromatography?

d) Cleavage by trypsin followed by gel-filtration chromatography gives the expected 6 products, which you sequence (shown in order of emergence from column): T-1 ETMESSAGEFGR T-2 SQTWALDHSECR T-4 TCR S-5 TME Think About It: T-5 NP e) Cleavage by Staph. aureus V8 protease followed by gel-filtration chromatography gives the expected 7 products, which you sequence (shown in order of emergence from column): S-1 RSQTWALD S-2 FGRGPQD S-3 NKTCRNP S-6 CRE T-3 GPQDNK S-7 HSE T-6 R S-4 SSAGE 1. Deduce the primary structure of this polypeptide. 2. Why would cyanogen bromide not be a good choice as a cleavage reagent? 3. Can you account for the order of elution of trypsin digest peptides from gel-filtration chromatography?

Biochemistry
Biochemistry
9th Edition
ISBN:9781319114671
Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Chapter1: Biochemistry: An Evolving Science
Section: Chapter Questions
Problem 1P
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2. You are in a South American rain forest looking for naturally occuring peptides with potential as drugs. You have a mobile biochemistry lab with common reagents and enzymes, an amino-acid analyzer, gel- filtration and ion-exchange chromatography, and electrophoresis. You also have an Edman Sequenator, but you have contaminated one or more of your reagents, and as a result, you cannot sequence peptides longer than about 12 residues before contaminants obscure the results. While screening extracts from the ovaries of an tropical orchid, you find a peptide with potential as an antiviral. Deduce its amino-acid sequence using the available tools. a) MW by electrophoresis can tell you how big a sequencing problem you are up against. Result: about 4000 b) Amino-acid analysis can help you decide how to fragment the peptide for sequencing: Result: A₂C₂D₂E4FG3HKLMN₂P2Q2R4S4T3W c) How many peptides expected from each of these possible cleavage reagents? Cyanogen bromide (C-side of M). Staph. aureus V8 protease (C-side of D and E). Trypsin (C-side of K and R). d) Cleavage by trypsin followed by gel-filtration chromatography gives the expected 6 products, which you sequence (shown in order of emergence from column): T-1 ETMESSAGEFGR T-2 SQTWALDHSECR T-4 TCR S-1 RSQTWALD S-5 TME e) Cleavage by Staph. aureus V8 protease followed by gel-filtration chromatography gives the expected 7 products, which you sequence (shown in order of emergence from column): Think About It: S-2 FGRGPQD T-5 NP S-6 CRE S-3 NKTCRNP T-3 GPQDNK S-7 HSE T-6 R S-4 SSAGE 1. Deduce the primary structure of this polypeptide. 2. Why would cyanogen bromide not be a good choice as a cleavage reagent? 3. Can you account for the order of elution of trypsin digest peptides from gel-filtration chromatography? 4. Predict the order of elution of the tryptic peptides from a cation-exchange column eluted with pH-8.5 buffer and a salt gradient. 5. Predict the order of elution of the V8 protease peptides from an anion exchange chromatography column eluted with a pH-6.5 buffer and a salt gradient. 6. For both sets of peptides, predict the order of elution from a hydrophobic interaction chromatography column, given that the hydrophobic amino acids are V,L,I,F,A, and M.
Transcribed Image Text:2. You are in a South American rain forest looking for naturally occuring peptides with potential as drugs. You have a mobile biochemistry lab with common reagents and enzymes, an amino-acid analyzer, gel- filtration and ion-exchange chromatography, and electrophoresis. You also have an Edman Sequenator, but you have contaminated one or more of your reagents, and as a result, you cannot sequence peptides longer than about 12 residues before contaminants obscure the results. While screening extracts from the ovaries of an tropical orchid, you find a peptide with potential as an antiviral. Deduce its amino-acid sequence using the available tools. a) MW by electrophoresis can tell you how big a sequencing problem you are up against. Result: about 4000 b) Amino-acid analysis can help you decide how to fragment the peptide for sequencing: Result: A₂C₂D₂E4FG3HKLMN₂P2Q2R4S4T3W c) How many peptides expected from each of these possible cleavage reagents? Cyanogen bromide (C-side of M). Staph. aureus V8 protease (C-side of D and E). Trypsin (C-side of K and R). d) Cleavage by trypsin followed by gel-filtration chromatography gives the expected 6 products, which you sequence (shown in order of emergence from column): T-1 ETMESSAGEFGR T-2 SQTWALDHSECR T-4 TCR S-1 RSQTWALD S-5 TME e) Cleavage by Staph. aureus V8 protease followed by gel-filtration chromatography gives the expected 7 products, which you sequence (shown in order of emergence from column): Think About It: S-2 FGRGPQD T-5 NP S-6 CRE S-3 NKTCRNP T-3 GPQDNK S-7 HSE T-6 R S-4 SSAGE 1. Deduce the primary structure of this polypeptide. 2. Why would cyanogen bromide not be a good choice as a cleavage reagent? 3. Can you account for the order of elution of trypsin digest peptides from gel-filtration chromatography? 4. Predict the order of elution of the tryptic peptides from a cation-exchange column eluted with pH-8.5 buffer and a salt gradient. 5. Predict the order of elution of the V8 protease peptides from an anion exchange chromatography column eluted with a pH-6.5 buffer and a salt gradient. 6. For both sets of peptides, predict the order of elution from a hydrophobic interaction chromatography column, given that the hydrophobic amino acids are V,L,I,F,A, and M.
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