This question has been posted previously with parts A B and C already answered. Please focus on only D, E and F Provided is the question alongside with the added materials next to it. Thank you
This question has been posted previously with parts A B and C already answered. Please focus on only D, E and F Provided is the question alongside with the added materials next to it. Thank you
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
Related questions
Question
This question has been posted previously with parts A B and C already answered. Please focus on only D, E and F Provided is the question alongside with the added materials next to it. Thank you
![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.
1) MW by electrophoresis can tell you how big a sequencing problem you are up against.
Result: about 4000
2) Amino-acid analysis can help you decide how to fragment the peptide for sequencing:
Result: A2C2D2E4FG3HKLMN2P2Q2R4S4T3W
3) 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).
4) 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-3 GPQDNK
T-4 TCR T-5 NP T-6 R
5) 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-4 SSAGE
S-5 TME S-6 CRE S-7 HSE
Think About It:
A. Deduce the primary structure of this polypeptide.
B. Why would cyanogen bromide not be a good choice as a cleavage reagent?
C. Can you account for the order of elution of trypsin digest peptides from gel-filtration chromatography?
D. Predict the order of elution of the tryptic peptides from a cation-exchange column eluted with pH-8.5
buffer and a salt gradient.
E. 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.
F. 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.](/v2/_next/image?url=https%3A%2F%2Fcontent.bartleby.com%2Fqna-images%2Fquestion%2Fb8a5a0f0-bcc2-4eba-9f62-efc6e1f3b408%2Fe3bef760-0e79-4298-93fb-a8cb7d0db63d%2Fnji8zro_processed.jpeg&w=3840&q=75)
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.
1) MW by electrophoresis can tell you how big a sequencing problem you are up against.
Result: about 4000
2) Amino-acid analysis can help you decide how to fragment the peptide for sequencing:
Result: A2C2D2E4FG3HKLMN2P2Q2R4S4T3W
3) 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).
4) 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-3 GPQDNK
T-4 TCR T-5 NP T-6 R
5) 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-4 SSAGE
S-5 TME S-6 CRE S-7 HSE
Think About It:
A. Deduce the primary structure of this polypeptide.
B. Why would cyanogen bromide not be a good choice as a cleavage reagent?
C. Can you account for the order of elution of trypsin digest peptides from gel-filtration chromatography?
D. Predict the order of elution of the tryptic peptides from a cation-exchange column eluted with pH-8.5
buffer and a salt gradient.
E. 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.
F. 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.
![Protein Sequencing Problems
To get you started, here is a table with the specificities for some of the enzymatic
and chemical procedures used in sequencing proteins:
Procedure
Edman Degradation
Carboxypeptidase A
Carboxypeptidase B
Cyanogen Bromide
aminoethylation
Trypsin
Chymotrypsin
Staphylococccus aureus V8
Protease
Anhydrous Cyanogen Bromide C side of R
Submaxillarus Protease
Endoproteinase Lys C
Asp-N-protease
Thermolysin
Pepsin
Acid Phosphatase
Acid Hydrolysis
Site
R₁-1
R₂
C side of N terminus R = any amino acid
N side of C terminus R, # Arg, Lys, Pro
R₁-1 Pro
N side of C terminus R Arg, Lys, AECys
R₁-1 # Pro
R = Met
C side of R₁
Cys residue
C side of R
Cside of R
C side of R
C side of R₁
C side of R
N side of R
N side of R₁
N side of R
Specificity
C side of R₁
Breaks all peptide
bonds
Rn+1
R = Met, Trp
Reacts with Cys to give
AECys
R = Lys, Arg, AECys
R₂+1 #Pro
R = Phe, Trp, Tyr, Leu
R₁+1 #Pro
R = Asp D, Glu E
R = Arg
R = Lys K
R = Asp D, Glu E
Comment
R=Leu, Asp, Glu, Phe,
Tyr, Trp
R₁+1 # Pro
R = Asp, Glu
Ineffective for blocked N termini
Removes 1 (usually up to 4) residues
sequentially
Removes 1 (usually up to 4) residues
sequentially
Highly Specific
Highly Specific
Highly Specific
Sometimes cleaves at other sites
R = Leu, Ile, Phe, Trp, Tyr, Sometimes cleaves at other sites
Val
R₁+1 #Pro
The table uses terms like "R". I've drawn a short piece of polypeptide so you
can visualize where these cleavages are taking place.
N side of R₁
Fairly nonspecific
Highly Specific
C side of R₁
If the amino acids are listed with dashes connecting them, they are in sequence.
If the amino acids are listed separated by commas, they are not in order.
For example.....
If I treat G-K-A-V-M with trypsin I will get two pieces. One will be (G-K) and
the other will be (A-V-M). If I only know the composition (and not the actual
sequence for these pieces) it would be written (G, K) and (A, M, V).](/v2/_next/image?url=https%3A%2F%2Fcontent.bartleby.com%2Fqna-images%2Fquestion%2Fb8a5a0f0-bcc2-4eba-9f62-efc6e1f3b408%2Fe3bef760-0e79-4298-93fb-a8cb7d0db63d%2F9yo3gje_processed.jpeg&w=3840&q=75)
Transcribed Image Text:Protein Sequencing Problems
To get you started, here is a table with the specificities for some of the enzymatic
and chemical procedures used in sequencing proteins:
Procedure
Edman Degradation
Carboxypeptidase A
Carboxypeptidase B
Cyanogen Bromide
aminoethylation
Trypsin
Chymotrypsin
Staphylococccus aureus V8
Protease
Anhydrous Cyanogen Bromide C side of R
Submaxillarus Protease
Endoproteinase Lys C
Asp-N-protease
Thermolysin
Pepsin
Acid Phosphatase
Acid Hydrolysis
Site
R₁-1
R₂
C side of N terminus R = any amino acid
N side of C terminus R, # Arg, Lys, Pro
R₁-1 Pro
N side of C terminus R Arg, Lys, AECys
R₁-1 # Pro
R = Met
C side of R₁
Cys residue
C side of R
Cside of R
C side of R
C side of R₁
C side of R
N side of R
N side of R₁
N side of R
Specificity
C side of R₁
Breaks all peptide
bonds
Rn+1
R = Met, Trp
Reacts with Cys to give
AECys
R = Lys, Arg, AECys
R₂+1 #Pro
R = Phe, Trp, Tyr, Leu
R₁+1 #Pro
R = Asp D, Glu E
R = Arg
R = Lys K
R = Asp D, Glu E
Comment
R=Leu, Asp, Glu, Phe,
Tyr, Trp
R₁+1 # Pro
R = Asp, Glu
Ineffective for blocked N termini
Removes 1 (usually up to 4) residues
sequentially
Removes 1 (usually up to 4) residues
sequentially
Highly Specific
Highly Specific
Highly Specific
Sometimes cleaves at other sites
R = Leu, Ile, Phe, Trp, Tyr, Sometimes cleaves at other sites
Val
R₁+1 #Pro
The table uses terms like "R". I've drawn a short piece of polypeptide so you
can visualize where these cleavages are taking place.
N side of R₁
Fairly nonspecific
Highly Specific
C side of R₁
If the amino acids are listed with dashes connecting them, they are in sequence.
If the amino acids are listed separated by commas, they are not in order.
For example.....
If I treat G-K-A-V-M with trypsin I will get two pieces. One will be (G-K) and
the other will be (A-V-M). If I only know the composition (and not the actual
sequence for these pieces) it would be written (G, K) and (A, M, V).
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