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-3 GPQDNK T-4 TCR T-5 NP T-6 R 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-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

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
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Please answer the following parts of the Biochemistry question while referencing the provided information. Thank you

**Educational Exercise: Determining Amino Acid Sequences from Peptide Cleavage**

**Scenario:**

You are in a South American rainforest searching for naturally occurring peptides with potential drug capabilities. Equipped with a mobile biochemistry lab, you have the tools for amino acid analysis, chromatography, and electrophoresis, but you face challenges with sequencing due to contaminated reagents. The focus is to deduce the amino-acid sequence of a peptide with antiviral potential using available techniques.

**Key Points:**

a) **Molecular Weight (MW) Determination via Electrophoresis:**

- Electrophoresis helps estimate the size of the sequencing challenge.
- Result: About 4000 daltons.

b) **Amino-Acid Analysis and Sequence Fragmentation:**

- Used to determine effective peptide fragmentation methods.
- Result: A2C2D2E4FG3HKLMN2P2Q2R4S4T3W

c) **Expected Peptides from Cleavage Reagents:**

- **Cyanogen Bromide**: Cleaves at the C-side of Methionine (M).
- **Staphylococcus aureus V8 Protease**: Cleaves at the C-side of Aspartic acid (D) and Glutamic acid (E).
- **Trypsin**: Cleaves at the C-side of Lysine (K) and Arginine (R).

d) **Trypsin Cleavage and Chromatographic Analysis:**

- Following trypsin cleavage, gel-filtration chromatography yields six products:
  - T-1: ETMESSAGEFGR
  - T-2: SQTWALDHSECR
  - T-3: GPQDNK
  - T-4: TCR
  - T-5: NP
  - T-6: R

e) **Staph. aureus V8 Protease Cleavage and Chromatographic Analysis:**

- Yields seven products upon gel-filtration:
  - S-1: RSQTWALD
  - S-2: FGRPGQD
  - S-3: NKTCRNP
  - S-4: SSAGE
  - S-5: TME
  - S-6: CRE
  - S-7: HSE

**Critical Thinking:**

A. **Deduction of Polypeptide Primary Structure:**
Transcribed Image Text:**Educational Exercise: Determining Amino Acid Sequences from Peptide Cleavage** **Scenario:** You are in a South American rainforest searching for naturally occurring peptides with potential drug capabilities. Equipped with a mobile biochemistry lab, you have the tools for amino acid analysis, chromatography, and electrophoresis, but you face challenges with sequencing due to contaminated reagents. The focus is to deduce the amino-acid sequence of a peptide with antiviral potential using available techniques. **Key Points:** a) **Molecular Weight (MW) Determination via Electrophoresis:** - Electrophoresis helps estimate the size of the sequencing challenge. - Result: About 4000 daltons. b) **Amino-Acid Analysis and Sequence Fragmentation:** - Used to determine effective peptide fragmentation methods. - Result: A2C2D2E4FG3HKLMN2P2Q2R4S4T3W c) **Expected Peptides from Cleavage Reagents:** - **Cyanogen Bromide**: Cleaves at the C-side of Methionine (M). - **Staphylococcus aureus V8 Protease**: Cleaves at the C-side of Aspartic acid (D) and Glutamic acid (E). - **Trypsin**: Cleaves at the C-side of Lysine (K) and Arginine (R). d) **Trypsin Cleavage and Chromatographic Analysis:** - Following trypsin cleavage, gel-filtration chromatography yields six products: - T-1: ETMESSAGEFGR - T-2: SQTWALDHSECR - T-3: GPQDNK - T-4: TCR - T-5: NP - T-6: R e) **Staph. aureus V8 Protease Cleavage and Chromatographic Analysis:** - Yields seven products upon gel-filtration: - S-1: RSQTWALD - S-2: FGRPGQD - S-3: NKTCRNP - S-4: SSAGE - S-5: TME - S-6: CRE - S-7: HSE **Critical Thinking:** A. **Deduction of Polypeptide Primary Structure:**
**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                       | Site                   | Specificity                                               | Comment                                       |
|---------------------------------|------------------------|-----------------------------------------------------------|-----------------------------------------------|
| Edman Degradation               | C side of N terminus   | Rₙ = any amino acid                                       | Ineffective for blocked N termini            |
| Carboxypeptidase A              | N side of C terminus   | Rₙ ≠ Arg, Lys, Pro<br>Rₙ₊₁ ≠ Pro                          | Removes 1 (usually up to 4) residues sequentially |
| Carboxypeptidase B              | N side of C terminus   | Rₙ ≠ Arg, Lys, AECys<br>Rₙ₊₁ ≠ Pro                        | Removes 1 (usually up to 4) residues sequentially |
| Cyanogen Bromide                | C side of Rₙ           | Rₙ = Met                                                  | Highly Specific                               |
| Anhydrous Cyanogen Bromide      | C side of Rₙ           | Rₙ = Met, Trp                                             | Highly Specific                               |
| Aminoethylation                 | Cys residue            | Reacts with Cys to give AECys                             |                                               |
| Trypsin                         | C side of Rₙ           | Rₙ = Lys, Arg, AECys<br>Rₙ₊₁ ≠ Pro                        | Highly Specific                               |
| Chymotrypsin                    | C side of Rₙ           | Rₙ = Phe, Trp, Tyr, Leu<br>Rₙ₊₁ ≠ Pro                     | Sometimes cleaves at other sites              |
| Staphylococcus aureus V8 Protease | C side of Rₙ         | Rₙ = Asp D, Glu E                                         |                                               |
| Submaxillarius Protease         | C side of Rₙ           | Rₙ = Arg                                                  |                                               |
| Endoproteinase Lys C             | C side of Rₙ           | Rₙ = Lys K                                                |                                               |
| Asp-N-protease                  | N side of Rₙ           | Rₙ = Asp D, Glu E                                         |                                               |
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 | Site | Specificity | Comment | |---------------------------------|------------------------|-----------------------------------------------------------|-----------------------------------------------| | Edman Degradation | C side of N terminus | Rₙ = any amino acid | Ineffective for blocked N termini | | Carboxypeptidase A | N side of C terminus | Rₙ ≠ Arg, Lys, Pro<br>Rₙ₊₁ ≠ Pro | Removes 1 (usually up to 4) residues sequentially | | Carboxypeptidase B | N side of C terminus | Rₙ ≠ Arg, Lys, AECys<br>Rₙ₊₁ ≠ Pro | Removes 1 (usually up to 4) residues sequentially | | Cyanogen Bromide | C side of Rₙ | Rₙ = Met | Highly Specific | | Anhydrous Cyanogen Bromide | C side of Rₙ | Rₙ = Met, Trp | Highly Specific | | Aminoethylation | Cys residue | Reacts with Cys to give AECys | | | Trypsin | C side of Rₙ | Rₙ = Lys, Arg, AECys<br>Rₙ₊₁ ≠ Pro | Highly Specific | | Chymotrypsin | C side of Rₙ | Rₙ = Phe, Trp, Tyr, Leu<br>Rₙ₊₁ ≠ Pro | Sometimes cleaves at other sites | | Staphylococcus aureus V8 Protease | C side of Rₙ | Rₙ = Asp D, Glu E | | | Submaxillarius Protease | C side of Rₙ | Rₙ = Arg | | | Endoproteinase Lys C | C side of Rₙ | Rₙ = Lys K | | | Asp-N-protease | N side of Rₙ | Rₙ = Asp D, Glu E | |
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