December 21, 2000 name______________________section___

Final Examination - 115:413 Experimental Biochemistry

Part A - multiple choice; answer each question by circling the letter of the correct answer. Each question is worth 2 points.

1. Which of the following is not a recommendation in the use of pipetters?
a. Delivering the tip contents onto the wall of the receiving tube.
b. Pushing the plunger down to the second stop to expel all contents.
c. Setting the volume above the stated maximum capacity of the pipetter.
d. Holding the pipetter vertical when sucking up solution into the tip.

2. The sample standard deviation is
a. Square root of (sum of deviations from the mean/no. of observations).
b. Square root of (sum of squares of deviations from the mean/no. of observations).
c. Square root of (sum of squares of deviations from the mean/no. of observations - 1).
d. Sum of squares of deviations from the mean/no. of observations.

3. 0.1 ml of stock solution is diluted with 2.4 ml H₂O. Fifty microliters of this solution is diluted with 0.45 ml H₂O. The overall dilution is
a. 1:240 b. 1:250 c. 1:400 d. 1:500

4. Adding one drop of 0.5 m KOH to an indicator-containing solution causes a color change from yellow to red and a pH change from 6.5 to 10. This suggests that
a. there is substantial buffer capacity in the solution.
b. there is substantial free H⁺ in the solution.
c. a pK_a of the indicator lies between 6.5 and 10.
d. all of the above.

5. The pK_as of glutamic acid are approximately 2.0, 4.0 and 10.0. What is the pH of an 0.1 M solution of monosodium glutamate?
a. 3.0 b. 4.0 c. 7.0 d. 10.0

6. The units of an extinction coefficient for a protein determination method would be
a. L·mole^-1cm^-1 b. M^-1cm^-1 c. mg·ml^-1cm^-1 d. ml·mg^-1cm^-1

7. A plot of absorbance vs. concentration in the Lowry method for protein determination has a negative y intercept. What is a likely explanation of this?
a. The blank has higher absorbance than it should (tube contaminated with protein?)
b. The color-producing reaction is incomplete.
c. Beer's law does not hold at low absorbance values.
d. The instrument was blanked on a solution with too low an absorbance.

8. Which method for protein determination is least subject to interference by non-protein compounds?
a. Coomassie Blue b. Lowry c. biuret d. ultraviolet abs.

9. A 1 mg/ml solution of lysozyme has about twice the A₂₈₀ of a 1 mg/ml solution of ovalbumin. This suggests that it has a higher content (than ovalbumin) of
a. arginine. b. cysteine. c. tryptophan. d. histidine.

10. One of the following will not react in the phenol-H₂SO₄ method:
a. maltose b. sucrose c. arabinose d. sorbitol

11. Optical rotation of a carbohydrate solution is proportional to all but one of these:
a. [a]_d b. concentration of the solution
c. length of the polarimeter tube d. number of chiral centers

12. Which method for carbohydrate determination is carried out at neutral pH?
a. orcinol b. indole c. glucose oxidation d. Nelson-Somogyi

13. After graduating you get a job with the Istituto Superiore de Sanità, an Italian government agency whose responsibilities include checking that spaghetti is made only with durum wheat (starch, glucose polymer). It is reported that one manufacturer is using instead flour from the Jerusalem artichoke root, which contains inulin, a fructose polymer ending with a glucose residue attached in 2Æ1 linkage, as in sucrose. Remembering your biochemistry lab experiments, you check this using
a. the indole reaction. b. the Nelson-Somogyi reaction.
c. the phenol-H₂SO₄ reaction. d. the glucose oxidase reaction.

14. The independently determined values in a protein purification table are
a. units/ml, mg protein/ml, yield b. volume, units/ml, mg protein/ml.
c. units/ml, mg protein/ml, specific activity d. units/ml, units/mg, yield

15. d-Amino acid oxidase is eluted from the DEAE-Sepharose column by increasing the concentration of
a. MOPS b. crotonate c. sodium d. chloride

16. The ratio A₄₆₀/A₂₈₀ of fractions from column chromatography of d-amino acid oxidase is a rough measure of their
a. specific activity b. enzymatic activity c. protein concentration d. purif. factor

17. Which of these procedures did you not use in purification of d-amino acid oxidase?
a. irreversible denaturation b. (NH₄)₂SO₄ precipitation
c. hydrophobic chromatography d. gel filtration
e. none (all were used)

18. Those who did experiment #11 saw that the rate of pyruvate production declined after 10 min even if catalase was present and the tubes were shaken. This indicates that the decline is not due to
a. using up the d-alanine in the solution. b. using up the oxygen in the solution.
c. inhibition by product peroxide. d. enzyme denaturation.

19. Most of you observed that the pH optimum of d-amino acid oxidase is quite high, ≈9.5. This suggests that the true substrate may be
a. alanine anion (with NH₂) b. alanine zwitterion (with NH₃⁺)
c. superoxide anion (O₂^-·) d. peroxide anion (HOO^-)

20. Inhibition of d-amino acid oxidase by benzoate is expected to be
a. competitive vs. d-alanine. b. noncompetitive vs. d-alanine.
c. uncompetitive vs. d-alanine. d. competitive vs. oxygen.

21. You have determined e₄₆₃ of your d-amino acid oxidase, using a molar concentration of the enzyme based on a Coomassie Blue protein determination, whiuch was based on a standard curve using bovine serum albumin. Later you find that bovine albumin gives 50% more color (A₅₉₅/A₄₆₆) per mg than an average protein. What is the likely effect of this on your molar extinction coefficient, as originally determined?
a. It is 33% low. b. It is 50% low. c. It is 50% high. d. none, because it is a molar extinction coefficient.

22. In a SDS polyacrylamide gel system, the SDS
a. associates with the peptide bonds.
b. disrupts the native secondary structure of the proteins.
c. disrupts the native tertiary structure of the proteins.
d. makes the proteins have a constant negative charge density.
e. all of the above.

23. The critical feature of the reservoir buffer in stacking gel electrophoresis is
a. its cation has a high pK_a, well above the pH of the stacking gel.
b. its anion has low mobility at the pH of the stacking gel.
c. its anion has high mobility at the pH of the running gel.
d. it dissipates heat generated during the run.

24. Our native gel assay for d-amino acid oxidase depends on what reaction?
a. Reduction of phenazine methosulfate by reduced FAD.
b. Oxidation of Nitro Blue tetrazolium by H₂O₂.
c. Reaction of ammonium ion with Nitro Blue tetrazolium.
d. Oxidation of phenazine methosulfate by d-amino acid oxidase.

25. Without which component acrylamide will polymerize, but not gel?
a. ammonium persulfate b. TEMED
c. oxygen d. N,N-methylenebisacrylamide

26. The enzyme-linked antibody in western blot visualization is an antibody to
a. rabbit immunoglobulin. b. alkaline phosphatase.
c. d-amino acid oxidase. d. Coomassie Blue.

27. Isoelectric focusing is particularly good at separating
a. different oligomers of the same protein.
b. isoforms of the same protein which differ in charge.
c. native and denatured forms of the same protein.
d. enzymes with the same activity but different molecular weight.

Part B - Short Answers.

1. (3 pts) Write the balanced chemical reaction for the reaction catalyzed by d-amino acid oxidase. Show structures, not just names.

2. (3 pts) What are (supposed) advantages of using A₅₉₅/A₄₆₆, rather than just A₅₉₅, for measuring protein with the Coomassie Blue reagent?

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

1. To a malonic acid solution, 100 ml of 0.05 M, is added 1.0 g Tris (tris[hydroxymethyl]aminomethane, mol. wt. 121, pK_a = 8.3). The pK₁ of malonic acid is 2.7, pK₂ is 5.7.

(1 pt) How many millimoles of malonic acid and Tris are present?

(1 pt) Which pK_a will you use to calculate the pH?

(3 pts) What is the pH?

2. (5 points) A bovine serum albumin standard solution, 10.0 mg/ml, gives the following results in the biuret method for protein determination:

ml BSA 0 0.2 0.4 0.6 1.0
A₅₄₅₄₀ 0 0.121 0.238 0.366 0.595

A solution of another protein gives the following results:

ml unknown 0 0.2 0.4 0.6 1.0
A₅₄₀ 0 0.082 0.158 0.241 0.398

Calculate the protein concentration of the unknown solution. Use graph below if desired.

3. (5 points) Observed R_ms and molecular weights of the standard proteins for molecular weight determination by SDS gel electrophoresis are as follows:

Protein R_m mol. wt. Protein R_m mol. wt.

aprotinin 0.88 6,500 ovalbumin 0.415 45,000
a-lactalbumin 0.69 14,200 albumin, bovine serum 0.32 66,000
trypsin inhibitor 0.61 20,000 b-galactosidase 0.186 116,000
carbonic anhydrase 0.52 29,000 myosin 0.05 205,000

An unknown protein has an R_m of 0.55 on the same gel. Calculate its molecular weight (use a graph below, or fit the standard molecular weights to an appropriate equation) .

4. The specific rotations of carbohydrates used in our experiment are:

Sugar [a]_d Sugar [a]_d Sugar [a]_d

d-glucose +52.8° l-fucose^@ -75.6° d-trehalose^∂ +178°

d-fructose# -92.0° l-rhamnose^@ +8.9° d-sucrose^∂# +66.5°

d-galactose +81.7° d-sorbitol (glucitol)^◊ -2.0° d-maltose^∂ +129.0°

l-sorbose# -42.7° l-arabinose* +104.0° d-lactose^∂ +52.3°

d-mannose +14.1° d-xylose* +18.6° d-cellobiose^∂ +34.5°

*Pentose ^#Ketose ^∂Disaccharide ^@6-deoxyhexose ^◊Sugar alcohol The others are aldohexoses.

The unknown (90 mg/ml, 2 dm path length) shows an optical rotation of +9.0°; also observed were +9.1° for d-glucose, -17.4° for d-fructose, +23.2° for d-maltose, +33.2° for d-trehalose, +13.3° for l-arabinose, +9.0° for lactose, +14.6° for galactose (all at 90 mg/ml, 2 dm).

The following data are determined in the colorimetric measurements of carbohydrates (data are shown for only one level of standard sugars):

Phenol-H₂SO₄: 0.4 ml glucose A₄₈₀ = 1.704, maltose 1.718, fructose 1.931, unknown 1.878.

Orcinol: 0.3 ml arabinose A₆₆₀ .= 0.557, glucose 0.047, fructose 0.195, unknown 0.098.

Indole: 0.3 ml fructose A₄₈₀ = 0.499, sucrose 0.262, glucose 0.058, lactose 0.054, unknown 0.061

Nelson-Somogyi: 0.4 ml glucose A₆₆₀ = 1.919, maltose 0.888, lactose 1.087, unknown 1.01.

Alk. ferricyanide: no sugar A₄₂₀ = 1.64, 1.0 ml glucose 0.721, lactose 1.09, maltose 1.37, sucrose 1.63, unknown 1.18.

Glucose oxidase: 1.0 ml glucose A₅₀₀ = 1.038, sucrose 0.034, lactose 0.003, unknown 0.002.

(5 pts) Identify the unknown sugar.

(5 pts) Explain its behavior, compared to standard sugars, in each test; does it or does it not react? Why? Which test tells you the most about its identity?

5. Below is a chart trace from a polarograph assay (volume 3 ml) of d-amino acid oxidase.

(2 pts) Draw a straight line and determine initial velocity (chart width/min or cm/min) of the reaction observed.

(3 pts) Calculate the activity of the stock enzyme in µmoles/min·ml stock. Amount and dilution of enzyme used are on the chart. Solubility of oxygen in water at 37° = 0.26 mM.

6. a) The following rates of enzymatic oxidation of samples of 0.02 m dl-norleucine, diluted to an assay volume of 3.0 ml, are observed in the peroxidase assay:

ml dl-norleu/PP_i 0.03 0.06 0.125 0.25 0.50 1.0 2.0

[d-norleu], mm:

∆A₅₀₀/min 0.083 0.143 0.227 0.312 0.385 0.435 0.465

a. (3 pts) Calculate D-norleucine concentration, and either [S]/v (Woolf plot) or 1/[S] and 1/v (Lineweaver-Burk plot) and plot on the graph below. The rate (v) is most easily plotted as ∆A₅₀₀/min. Label the axes clearly. e₅₀₀ of peroxidase product = 14,250 L/mole·cm.

1/[S]

1/v

[S]/v

b. (5 pts) Determine V_max (in whatever units you used above) and K_m (mM) from the data above.

c. (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?

d. (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?