4. 3. A novel octapeptide is isolated from the skin of a poisonous frog. The biotechnology...

  

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4. 3. A novel octapeptide is isolated from the skin of a poisonous frog. The biotechnology firm you work for gives you the following data and asks you to determine the sequence so that larger quantities can be made by peptide synthesis and used as an insecticide. For full credit: Explain the function of each reagent, enzyme, procedure and the results at each of steps 1-7. Be thorough and explain your reasoning. (Each part is 2 of 18 pts. and I want something written for each point even if obvious) 1. The amino acid composition following: 1. treatment with DTT and 2. acid hydrolysis of 1 nmole of the peptide gives the following results. Amino acid Ala Ser Lys Phe Val Glu Pro Cys 2. nmoles 0.92 0.73 0.97 0.88 0.99 0.81 1.01 0.92 What is your interpretation of this data? (2 pts.) and what did Step 1 and 2 do? Estimate the MW of the octapeptide (to nearest 100) (2 of 18 pts.) How did you get this number? Separate samples of reduced peptide are used in each of Steps 2-7: After treatment with dansyl chloride at pH 8 followed by acid hydrolysis, you obtain an odd colored derivative that is inconclusive. When you react a fresh sample with ninhydrin however, you obtain a nice brownish yellow derivative that you immediately recognize is due to the amino acid Explain. Da After treatment with carboxypeptidase, you obtain free Ser, plus a little Val. What does enzyme do? What is your conclusion and why? The peptide has weak UV absorbance at 280 nm. Interpret. 5 7. If you treat it with staphylococcal protease (V8), you get 2 fragments. After purification and Edman sequencing, you learn one fragment has sequence Ala- Val-Ser and the other has the sequence Pro-Lys-Cys-Phe-X. (What does V8 do?) Trypsin treatment gives 2 fragments, the smaller has the sequence Pro-Lys Explain, Interpret, What does trypsin do? After treatment with chymotrypsin, you get 2 fragments. One fragment has the sequence Pro-Lys-Cys-Phe. The other was found to have the partial sequence Glu-X-X-X. What does chymotrypsin do? 8. What is the sequence of the intact octapeptide? 9. (7 pts.) Critical Thinking Question A. After running an HPLC size exclusion column (SEC-HPLC) on a peptide sample fresh from the frog, you obtain an apparent MW of 2 kDa (-2,000 daltons). How does this compare to what you estimated from the sequence? What is a possible explanation for this observation? (2/7 pts.) B. How could you test this hypothesis? What techniques or reagents could you use? Describe a brief set of procedures to use. (5/7 pts.) 4. 3. A novel octapeptide is isolated from the skin of a poisonous frog. The biotechnology firm you work for gives you the following data and asks you to determine the sequence so that larger quantities can be made by peptide synthesis and used as an insecticide. For full credit: Explain the function of each reagent, enzyme, procedure and the results at each of steps 1-7. Be thorough and explain your reasoning. (Each part is 2 of 18 pts. and I want something written for each point even if obvious) 1. The amino acid composition following: 1. treatment with DTT and 2. acid hydrolysis of 1 nmole of the peptide gives the following results. Amino acid Ala Ser Lys Phe Val Glu Pro Cys 2. nmoles 0.92 0.73 0.97 0.88 0.99 0.81 1.01 0.92 What is your interpretation of this data? (2 pts.) and what did Step 1 and 2 do? Estimate the MW of the octapeptide (to nearest 100) (2 of 18 pts.) How did you get this number? Separate samples of reduced peptide are used in each of Steps 2-7: After treatment with dansyl chloride at pH 8 followed by acid hydrolysis, you obtain an odd colored derivative that is inconclusive. When you react a fresh sample with ninhydrin however, you obtain a nice brownish yellow derivative that you immediately recognize is due to the amino acid Explain. Da After treatment with carboxypeptidase, you obtain free Ser, plus a little Val. What does enzyme do? What is your conclusion and why? The peptide has weak UV absorbance at 280 nm. Interpret. 5 7. If you treat it with staphylococcal protease (V8), you get 2 fragments. After purification and Edman sequencing, you learn one fragment has sequence Ala- Val-Ser and the other has the sequence Pro-Lys-Cys-Phe-X. (What does V8 do?) Trypsin treatment gives 2 fragments, the smaller has the sequence Pro-Lys Explain, Interpret, What does trypsin do? After treatment with chymotrypsin, you get 2 fragments. One fragment has the sequence Pro-Lys-Cys-Phe. The other was found to have the partial sequence Glu-X-X-X. What does chymotrypsin do? 8. What is the sequence of the intact octapeptide? 9. (7 pts.) Critical Thinking Question A. After running an HPLC size exclusion column (SEC-HPLC) on a peptide sample fresh from the frog, you obtain an apparent MW of 2 kDa (-2,000 daltons). How does this compare to what you estimated from the sequence? What is a possible explanation for this observation? (2/7 pts.) B. How could you test this hypothesis? What techniques or reagents could you use? Describe a brief set of procedures to use. (5/7 pts.)

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To estimate the molecular weight MW of the octapeptide we need to calculate the sum of the MWs of the individual amino acids based on their nmoles The MWs for the amino acids are approximately as foll... View the full answer