December 14, 1993 name_________________________section___

Final Examination — 115:413 Experimental Biochemistry

Part A - multiple choice; answer each question by circling the letter of the correct answer.

Some of these questions are purely factual, some call for thinking.

1. Which of the following is not a term in the Henderson-Hasselbalch equation for pH of a buffer solution?
a. the concentration of the acidic form.
b. the concentration of the basic form.
c. the total concentration of the buffer.
d. the pK_a.

2. You are titrating a buffer with an indicator present. The indicator is blue in its basic form, yellow in its acidic form. The pH at which the indicator is green is
a. the pK_a of the buffer. b. the pK_a of the indicator.
c. the pI of the buffer. d. the pI of the indicator.

3. The equation for molar extinction coefficient is
a. e = A/cl b. e = cl/A c. e = Ac/l d. e = Al/c

4. Which method for protein determination is most sensitive?
a. Lowry b. biuret c. Coomassie Blue d. ultraviolet abs.

5. One tenth of a milliliter of a stock protein solution is diluted to 5 ml with water. Three tenths of a milliliter of this diluted solution is found to contain 0.018 mg protein. What is the protein concentration of the stock solution?
a. 18 mg/ml b. 0.6 mg/ml c. 6 mg/ml d. 3 mg/ml

6. "Setting the blank" on the spectrophotometer
a. means setting the instrument to read 0 with the reagents present, but not the sub-
stance being measured.
b. subtracts out the absorbance of the reagents.
c. is an example of running a control.
d. all of the above.

7. An optical density (≈ absorbance) of 2 corresponds to what % transmission?
a. 0.1 b. 1 c. 2 d. 10

8. The first step in enzyme purification is generally
a. extraction of the tissue from which it is derived.
b. (NH₄)₂SO₄ precipitation.
c. acidification to the enzyme's isoelectric point.
d. purification of the organelle in which it is found.

9. Enzyme assays (for measurement of amount of enzyme present) generally measure the amount of a product formed because
a. products can be measured more sensitively than protein.
b. many molecules of product are formed per molecule of enzyme.
c. disappearance of the substrate is less specific.
d. the Michaelis-Menten equation is derived for initial velocity conditions.

10. In a purification table, yield generally means
a. specific activity at a given step/specific activity at the previous step.
b. specific activity at a given step/specific activity at the first step.
c. total units at a given step/total units at the previous step.
d. total units at a given step/total units at the first step.

11. All but one of the following statements about (NH₄)₂SO₄ precipitation of proteins is true:
a. proteins precipitate at lowest (NH₄)₂SO₄ concentration when isoionic (at their pI).
b. the effectiveness of (NH₄)₂SO₄ precipitation is not affected by the total protein conc.
c. solubility of a protein depends on salt concentration by log s = A - m(salt conc.).
d. (NH₄)₂SO₄ is particularly effective because of its SO₄⁼ ion: ionic strength is propor-
tional to the square of charge of the ions.

12. The pyruvate assay is more sensitive than the peroxidase and polarograph assays primarily because
a. the extinction coefficient of the measured product is higher.
b. pyruvate is a direct product of the enzyme reaction.
c. the incubation time is longer.
d. it is a coupled assay.

13. Gel filtration separates proteins from smaller molecules by
a. excluding the protein molecules from pores in the gel particles.
b. excluding the small molecules from pores in the gel particles.
c. adsorbing the small molecules on the gel.
d. filtering the small molecules out of the solution.

14. The A₂₈₀ of a solution of pure d-amino acid oxidase is about what times the A₄₆₀ (assuming no benzoate present)?
a. 20x b. 8x c. 2x d. 1x

15. What kind of inhibitor of d-amino acid oxidase do you expect crotonate to be?
a. noncompetitive (vs. amino acid) b. uncompetitive (vs. amino acid)
c. competitive (vs. amino acid) d. competitive (vs. O₂)

16. Sodium dodecyl sulfate does the following in gel electrophoresis of proteins:
a. it denatures them, so that shape is not a factor in separation.
b. it separates the subunits of a protein made up of several subunits.
c. it associates with the peptide backbone, making all proteins negatively charged.
d. it overwhelms the charge on the side chains, so that all proteins have about the same
charge density.
e. all of the above.

17. In our electrophoretic system, the ion with the lowest mobility in the stacking gel is
a. the protein. b. the tracking dye. c. TrisH⁺. d. glycine. e. Cl^-.

18. Mobility in an SDS polyacrylamide gel is
a. inversely proportional to molecular weight b. directly proportional to mol. weight
c. inversely proportional to log mol. wt. d. directly proportional to log mol. wt.

19. Color reactions of carbohydrates in strong acid depend on what reaction?
a. oxidation of the aldehyde or ketone group b. opening of the furanose ring
c. dehydration to furfurals d. all of the above

20. A sugar solution does not react in the Nelson-Somogyi method and is dextrorotatory in the polarimeter. Hydrolysis with b-galactosidase makes the solution reactive in the Nelson-Somogyi method and levorotatory. In the indole method the sugar is about as reactive per mg as sucrose. A likely structure for the sugar is
a. b-galactosyl-(1->4)glucose.
b. b-fructosyl-(2->4)galactose.
c. b-galactosyl-(1->1)fructose..
d. b-galactosyl-(1->2)fructose.

21. After graduation you take a job with the Florida Orange Commission, checking the quality of Florida orange juice. One assignment is to check how much orange pulp is present in "country-style" juice, by determining the amount of pectin (found in pulp rather than free juice). To do this you might use what method?
a. carbazole b. orcinol c. indole d. ferricyanide

22. What sugar would react in the Nelson-Somogyi reaction, but not in the phenol-H₂SO₄ reaction?
a. glyceraldehyde b. sucrose c. sorbitol d. arabinose

23. Which assay would most readily distinguish between solutions of glucose and lactose?
a. phenol-H₂SO₄ b. polarimetry c. Nelson-Somogyi d. glucose oxidase

24. What is the function of chloroform in preparation of nucleic acids?
a. to denature proteins b. to extract pigments from the H₂O phase
c. to extract phenol from the H₂O phase d. to precipitate the nucleic acids
e. all of the above

25. What is the biggest problem in preparation of full-length mRNA (for cDNA synthesis)?
a. denaturation by phenol b. cleavage by ribonuclease
c. separation from proteins d. hydrolysis by base

26. Preparation of hydrolyzed RNA for column chromatography requires
a. neutralization of the base b. lowering the ionic strength
c. removal of particulate material d. all of the above

27. The nucleotides are eluted from the DEAE-Sephadex by competing
a. Cl^- b. b-alanine^± c. b-alanineH⁺ d. K⁺

28. A logarithmic gradient elution of the ribonucleotides might separate better
a. CMP and AMP. b. AMP and UMP. c. UMP and GMP. d. all the nucleotides.

29. If the true substrate of d-amino acid oxidase is the anion (d-alanine^-; pK₂ = 9.8), you would expect the K_m, expressed as concentration of total d-alanine to be
a. higher at pH 9.3 than at pH 8.3 b. lower at pH 9.3 than at pH 8.3
c. the same at pH 9.3 as at pH 8.3 d. cannot tell from the data given

30. Observation that all the assays of d-amino acid oxidase in the indophenol assay resulted in A₆₁₀ >1.0 suggests that
a. there was free NH₄⁺ in the dl-alanine substrate.
b. there was free NH₄⁺ in the enzyme preparation.
c. the enzyme is more active in this assay.
d. you made a mistake in setting up the assay.

31. EXTRA CREDIT: The pyruvate assay frequently shows a higher apparent activity in the crude extract than the peroxidase assay. Presence of which of these enzymes in the crude extract, removed at the (NH₄)₂SO₄ precipitation step, could explain this?
a. catalase b. l-amino acid oxidase c. pyruvate kinase d. lactate oxidase
For another two points, explain briefly why only your answer is correct.

Part B - Problems. Show all work and indicate your answer clearly.

1. Upper reservoir buffer for gel electrophoresis is prepared by adding solid Tris base (pK_a of TrisH⁺ = 8.3; mol. wt. = 121) to one liter of 0.192 m glycine (pK₂ = 9.87) until the pH reaches 8.8. (The Henderson-Hasselbalch equation must be satisfied for both Tris and glycine at this pH.)

a) Calculate the concentration of glycine anion at this pH. (4 pts)

b) Calculate how much total Tris base was added to reach this pH. (6 pts) (Hint: What is the concentration of TrisH⁺ equal to?)

2. The [a]_d of d-sucrose is +66.5°, of d-glucose is +52.8°, of d-fructose is -92.0°. The molecular weights are 342 g/mole for sucrose, 180 for glucose and fructose.
a) What will be the optical rotation of an 0.5 m solution of sucrose in a 20 cm cell? (3 pts)

b) What will be the optical rotation of the solution if the sucrose is completely hydrolyzed to glucose + fructose?

3. The millimolar extinction coefficients (units: ml/mmole^.cm) for CMP and AMP are:

e₂₆₀ e₂₈₀ e₂₆₀/e₂₈₀

CMP 6.2 13.0 0.48

AMP 14.7 3.3 4.5

A solution containing CMP and AMP has A₂₆₀ = 0.960, A₂₈₀ = 0.912. Calculate the molar concentration of the two nucleotides present. (6 pts)

4. (a) The following A₅₆₀ were observed for aliquots of a standard pyruvate solution in the dinitrophenylhydrazone assay:

mmoles 0 0.05 0.10 0.15 0.20 0.25
A₅₆₀ 0 0.176 0.347 0.525 0.701 0.875

Calculate the slope (factor to convert observed A₅₆₀ to mmoles pyruvate.) (2 pts) Use the graph if you wish.

(b) Calculate the molar extinction coefficient of pyruvate dinitrophenylhydrazone at 560 nm (assuming the same procedure used in the lab) (2 pts).

(c) The following A₅₆₀ values were observed in assay of samples of a 1:400 dilution of purified d-amino acid oxidase:

ml enzyme 0.05 0.1 0.2
A₅₆₀ 0.085 0.169 0.342

Using the standard curve in (a), calculate the mmoles pyruvate formed in each sample. (2 pts)

(d) From these results, calculate an average value for activity (mmoles pyruvate/min^.ml) of the stock enzyme solution. (2 pts)

5. a) The following rates of enzymatic oxidation of samples of 0.04 m d-norvaline, diluted to an assay volume of 3.0 ml, are observed in the peroxidase assay:

ml d-norvaline 0.03 0.06 0.12 0.24 0.50 1.0 1.5

[d-norvaline], mm:

∆A₅₀₀/min 0.091 0.158 0.250 0.353 0.444 0.486 0.511

Calculate the norvaline concentrations, the K_m for this substrate, and V_max in mmoles/ min for this amount of enzyme (hint for speed: calculate in A₅₆₀, then convert V_max to mmoles/min [e₅₀₀ of peroxidase product = 14,250]. Use graph below if desired.) (8 pts)

(b)(1 pt) If the enzyme used was 0.1 ml of a 1:20 dilution, what is the V_max in mmoles/min^. stock enzyme?

(c)(1 pt) If the stock enzyme had a protein concentration of 0.8 mg/ml, and the molecular weight (per subunit) is 50,000, what is the turnover number?