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Biochemistry of Metabolism (4650) Exam 1 Name _____________Key___________________ Answer all questions completely.

Show all work for full credit. 09-14-12

(3pts) 1. Cytochalasin D is a compound that disrupts microfilaments. Eukaryotic cells that were exposed to the drug had (Ref: Biol Cell. 1995;83(2-3):149-61) a. decreased ability to degrade biomolecules. c. altered cell shape. b. did not synthesize proteins efficiently. d. greater production of cellular DNA. (3pts) 2. The antibiotic tetracycline inhibits protein synthesis so it most likely directly acts on the a. ribosomes. c. Golgi. b. smooth endoplasmic reticulum. d. plasma membrane. (3pts) 3. Reaction 1: Go = -6.7 kJ/mol for A + B > C + D Reaction 2: Go = -3.2 kJ/mol for R + Q > S + T. Which reaction has a greater proportion of products at equilibrium? a. Reaction 1. c. They have the same proportion of products. b. Reaction 2. d. Cant say; more information is needed. (3pts) 4. Which reaction above proceeds more rapidly to achieve equilibrium? a. Reaction 1. c. They have the same reaction rate. b. Reaction 2. d. Cant say; more information is needed. (22pts) 5.a. Draw the predominate form of the dipeptide tyrosylglutamate as it appears at pH = 4.8.

b. Draw one hydrogen bond between your dipeptide and water. c. Circle all chiral carbons. d. Write the 3 letter code for tyrosine ____Tyr_____ and glutamate _______Glu______ e. The pI of this dipeptide is (circle one):a. 3.3 b. 6.3 c. 6.7 d. 7.2 e. 9.6 f. What is/are the buffering region(s) for this dipeptide? 1.2-3.2, 3.3-4.3, 8.1-11.1 (8.1-10.1 & 9.1-11.1) g. At pH 7.0, determine the ratio of the deprotonated side chain group of glutamate to the protonated form. Show your work and put your answer on the line below. [A-]/[HA] = 501:1 pH = pKa + log([A-]/[HA]) > 7.0 = 4.3 + log([A-]/[HA]) > 501 = [A-]/[HA]

page 2 (3pts) 6. Two solutions are separated by a semi-permeable membrane. Solution 1 contains 1.00 M KCl, while Solution 2 contains 0.25 M KCl. Due to osmosis, over time a. water moves from solution 1 to solution 2. c. KCl moves from solution 1 to solution 2. c. water moves from solution 2 to solution 1. c. KCl moves from solution 2 to solution 1.

(3pts) 7. Circle the one answer below that has to be true for a spontaneous reaction. Go <0 TS > H G >0 G < w

(9pts) 8.a. Anion exchange chromatography at pH = 6.3 is used to separate cytochrome c, myoglobin, ribonuclease A and serum albumen. Under these conditions which protein elutes first ______cytochrome c_________ last _______serum albumen______________

b. The best way to elute the protein from the column above is to add a solution containing a. sodium dodecyl sulfate. c. high ligand concentration. b. high salt concentration. d. organic solvent. c. If you did gel filtration chromatography instead to separate the proteins, which protein would elute first? serum albumen d. Circle the ion exchange resin you would choose to do anion exchange chromatography: carboxymethylcellulose or diethylaminoethyl sephadex (cellulose and sephadex are the inert parts of the resin) (4pts) 9. To the right are an SDS-PAGE gel and an isoelectric focusing gel. Each gel was used to separate lysozyme from serum albumen . In each gel, label the bands representing lysozyme (L) and serum albumen (SA). SDS-PAGE focusing Isoelectric

(2pts) 10. Affinity chromatography separates on the basis of: charge size hydrophobicity ability to bind a specific molecule (3pts) 11. Recognizable, regular local structure within a polypeptide chain is its (primary / secondary / tertiary / quaternary) structure. (Circle the one correct answer.)

(3pts) 12. Briefly explain why rotation around a peptide bond is restricted. Be as specific as possible. The peptide bond (C-N bond) has partial double bond character which prevents free rotation.

Name ________________Key___________________________________ (4pts) 13. Write the three letter code for: a. two non-polar amino acids. many choices

page 3

b. two cationic amino acids.

Lys, Arg, His (any 2)

(4pts) 14. Concisely but completely define hydrophobic interactions. Hydrophobic interactions are the association of non-polar molecules (or groups) to minimize their contact with water. (This occurs to maximize entropy because water molecules form highly organized clathrate structures around hydrophobic groups.)

(2pts) 15. When making synthetic peptides, you need to activate amino acids that you are adding to the peptide chain. Activation refers to making the a. alpha amine more reactive. c. alpha carboxylic acid more reactive. b. side chain more reactive. d. peptide bond more reactive. (3pts) 16. Which one of the following is least likely to form an -helix? a. polyglutamate at pH = 2.5 c. polyvaline at pH = 10.1 b. polyhistidine at pH = 4.5 d. polytyrosine at pH = 8.7 (3pts) 17. Every -helix is stabilized by hydrogen bonding between what groups? Be as specific as possible. C=O of amino acid n and H-N of amino acid n + 4 (2pts) 18. In -sheet structures, the side chains a. are in the plain of the sheet. b. point above the sheet.

c. point below the sheet. d. point above and below the sheet. -keratin below ~2 ~7 silk fibroin above ~9

(2pts) 19. Reducing agents have the greatest effect on the structure of: collagen (8pts) 20.a. In step one of Edman degradation would you want the pH to be:

b. Show the mechanism of the first step in Edman degradation, including the product of the first step. Use arrows to represent the movement of electrons (electron pushing). c. Briefly explain why you chose the pH you circled in part a, including how it relates to the mechanism.

The first step is done at high pH to deprotonate the N-terminal amine so it has a free pair of electrons for nucleophilic attack.

page 4 (2pts) 21. Draw an arrow showing each place trypsin can cut the peptide below.

Val-Pro-Ser-Lys

-Leu-Leu-Ile-Glu-Cys-Arg

-Phe-Trp-Met

(3pts) 22. Circle the graph below which best represents the effect of salt on the solubility of proteins.

(3pts) 23. Identify each of the partial sequences below as coming from collagen, -keratin, or silk fibroin a. -Gly-Pro-Val-Gly-Pro-Hyp-Gly-Ser-Pro-Gly-Pro-Hyp- ____collagen______________ b. -Gly-Ser-Gly-Ala-Gly-Ser-Gly-Ser-Gly-Ala-Gly-Ser- _____silk fibroin____________ c. -Leu-His-Val-Cys-Ala-Ser-Ile-Glu-Val-Tyr-Cys-Thr- _____-keratin_____________ (3pts) 24. The purpose of the acrylamide in SDS-PAGE is a. to give proteins a uniform charge to mass ratio. b. polymerize to form the gel network. c. carry current within the gel. d. provide the pH difference between the stacking and separation portions of the gel.

Selected Protein Data Protein cytochrome c fibrinogen hemoglobin lysozyme myoglobin ovalbumin pepsin 34,500 ribonuclease A serum albumin (human) 4.9 7.8 69,000 12,600 pI 10.6 5.8 7.1 11.0 7.0 4.6 <1.0 MW 13,400 340,000 64,500 14,400 16,900 45,000

pKA Values for the 20 Standard Amino Acids Amino Acid Ala Arg Asn Asp Cys 8.3 Glu Gln Gly His Ile Leu Lys Met Phe Pro Ser 4.3 2.2 2.3 6.0 2.4 2.4 10.5 2.3 1.8 2.1 9.2 9.1 10.6 2.2 2.2 9.1 9.6 1.8 9.7 9.6 2.2 9.7 -COOH Side Chain 2.4 12.5 2.0 3.9 1.7 10.8 8.8 2.1 9.8 -NH3+ 9.7 2.2 9.0

9.2

9.0

9.2

~13 Thr ~13 Trp Tyr Val 2.4 10.1 2.3 9.6 9.4 2.2 9.1 2.6 10.4

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