December 17, 1997 name______________________section___

Final Examination - 115:413 Experimental Biochemistry - Master

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 recommended in the use of micropipettors?
√a. Adjusting the pipettor above its maximum volume setting.
b. Pushing the plunger down to the first stop to suck up solution.
c. Giving the solution time to rise in the pipet tip.
d. Delivering onto the wall of the receiving tube, above the solution already in it.

2. Which of the following procedures would achieve a 1:400 dilution?
a. Ten microliters of stock solution + 40 microliters water, 10 microliters of this solution (the 10 + 40) + 40 microliters water.
b. Ten microliters of stock solution + 30 microliters water, 10 microliters of this solution + 30 microliters of water.
√c. Ten microliters of stock solution + 30 microliters of water, 10 microliters of this solution + 990 microliters of water.
d. Ten microliters of stock solution + 90 microliters of water, 10 microliters of this solution + 490 microliters of water.

3. When a biochemical reaction produces acid in a buffered solution,
a. no change takes place in the buffer.
√b. a small amount of the basic form of the buffer is converted to the acidic form.
c. a small amount of the acidic form of the buffer is converted to the basic form.
d. the basic form of the buffer is consumed.

4. If the second pK_a of H₂SO₄ is 1.9, the pK_a of ammonia (NH₄⁺ Æ NH₃ + H⁺) is 9.3, what will the pH of an 0.1 M (NH₄)₂SO₄ solution be (approximately)?
a. 1.9 √b. 5.6 c. 7.0 9.3

5. One of the following could not be units of an extinction coefficient:
a. L·g^-1cm^-1 b. ml·µmole^-1cm^-1 c. %^-1in^-1 √d. L·mole^-1cm

6. Which protein reagent has the highest absorbance itself at the wavelength at which you measure its reaction with protein?
a. biuret b. Lowry √c. Coomassie Blue d. A₂₈₀

7. Which of the following is not an advantage of the Zor & Seliger A₅₉₅/A₄₆₆ Coomassie Blue method?
a. the 'standard curve' is straighter, particularly at low net absorbance.
b. the slope of the standard curve per mg protein is larger.
c. the standard curve is linear to higher protein concentration.
√d. reading at two wave lengths takes longer.

8. An 'unknown substance' has high UV absorbance and reaction in the Lowry method, but does not react with Coomassie Blue or biuret reagent. It may be
√a. tyrosine b. cysteine c. arginine d. triethanolamine.

9. Dimethylaminoazobenzene isothiocyanate is used as a reagent in Edman degradation of proteins because
a. it reacts rapidly and completely with the N-terminal amino acid.
√b. its product from proteins has high absorbance of visible light.
c. it reacts with lysine e-NH₂ groups.
d. all of the above.

10. Even though the DNA sequence of a gene is now easier to determine than the amino acid sequence of the protein coded for by the gene, the latter is still important to do because
a. cDNA sequences are sought using oligonucleotide probes based on protein sequence.
b. one wants to know what N-terminal sequence remains after proteolytic processing.
c. the protein sequence tells you whether you have the right DNA sequence.
√d. all of the above.

11. When you extract the carboxypeptidase-digested sample with 70% ethanol, you are
a. denaturing the sample protein.
b. denaturing the carboxypeptidase.
√c. separating the released amino acids from the undigested protein.
d. removing the NH₄HCO₃ in which the carboxypeptidase was dissolved.

12. At what R_f are similar compounds best separated?
a. 0.9 √b. 0.5 c. 0.1 d. any

13. What would you expect the R_f of denatured protein to be in the chromatography of free amino acids?
√a. 0 b. 0.5 c. 0.9 d. can't tell

14. The first step in purification of d-amino acid oxidase uses all but one of the following processes:
√a. salting out b. heat denaturation c. acid denaturation d. isoelectric precipitation.

15. d-Amino acid oxidase is effectively purified by hydroxylapatite chromatography because
a. of those proteins present, it is the one most tightly bound to hydroxylapatite.
√b. of those proteins present, it is the one most loosely bound to hydroxylapatite.
c. it has many covalently bound phosphate groups on its surface.
d. its crystal structure is complementary to that of hydroxylapatite.

16. The final step in the purification procedure we used this year utilizes
a. charge interaction. b. isoelectric precipitation
√c. hydrophobic interaction d. gel filtration.

17. The most likely reason why activity in the pyruvate assay typically levels off at a final A₅₆₀ around 1.0 is
√a. oxygen depletion. b. enzyme denaturation
c. enzyme saturation. d. enzyme is in excess over substrate.

18. Which procedure would allow you to determine the K_m of d-amino acid oxidase for O₂?
a. Run polarograph assays at different enzyme concentrations; plot 1/v vs. 1/[E].
b. Run polarograph assays at different dl-alanine concentrations; plot 1/v vs 1/[d-ala].
√c. Run a single polarograph assay; determine rate over various segments of the assay curve, plot 1/v vs. 1/midpoint of the chart segment.
d. Run polarograph assays; determine how long it takes to reach 50% saturation, plot 1/time to reach 50% saturation vs. 1/[d-alanine].

19. The molar extinction coefficient of the pyruvate product is calculated by multiplying the slope of A₅₆₀ vs. µmole pyruvate by
a. 3 ml √b. 3000 ml c. 3x10⁶ ml d. 3x10⁹ ml.

20. The product of d-amino acid oxidase action of d-valine which is actually measured is
a. a-ketoisovalerate. b. pyruvate dinitrophenylhydrazone.
c. pyruvate. √d. a-ketoisovalerate dinitrophenylhydrazone.

21. On the basis of what volume should the substrate concentration in the pyruvate assay be calculated?
a. 0.5 ml. b. 0.6 ml. √c. 1.0 ml. d. 3.0 ml.

22. Na benzoate (mol. wt. 144), 0.1%, is diluted 0.2 ml Æ 3.45 ml. 0.05 ml and 0.2 ml of the diluted solution are used in pyruvate assays. What are the concentrations of benzoate in the assays?
a. 0.01, 0.04 mM √b. 0.02, 0.08 mM c. 0.05, 0.2 mM d. 0.1, 0.4 mM.

23. The absorbance of d-amino acid oxidase at 460 nm is due to
√a. bound FAD. b. bound benzoate. c. bound substrate. d. tyr and trp.

24. Which ionized side chains will be primarily responsible for movement of proteins in native gel electrophoresis?
a. tyrosine and tryptophan b. lysine and arginine
c. asparagine and glutamine √d. aspartic and glutamic acids

25. Tricine or glycine or other such compound in the reservoir buffer should
a. buffer at the pH of the reservoir buffer.
b. be mostly zwitterionic at the pH of the reservoir buffer.
√c. be mostly zwitterionic at the pH of the stacking gel buffer.
d. be mostly zwitterionic at the pH of the running gel buffer.

26. Sodium dodecyl sulfate in gel electrophoresis of proteins
a. associates with the peptide backbone, making all proteins negatively charged.
b. causes all proteins to have about the same charge density.
c. denatures them, so that shape is not a factor in separation.
√d. all of the above.

27. The role of ammonium persulfate in preparing the stacking gel is to
a. buffer the gel mixture. √b. initiate polymerization.
c. precipitate proteins. d. all of the above.

28. The native gel and western blot assays share one substrate:
a. d-alanine b. oxygen c. pyruvate √d. Nitro Blue tetrazolium

29. Proteins are transferred from electrophoresis gels to membranes ('western blots') primarily so that
√a. they are accessible to antibodies. b. they can be stained with Coomassie Blue.
c. they can react with substrates. d. they can bind nucleic acids.

30. In most of our native gel experiments, d-amino acid oxidase gave a reaction band in neither the gel assay nor the visualization of the western blot (but the blot of the SDS gel did react). From this you might conclude (think critically!) that
a. the enzyme was inactive. b. the antibody did not react with it.
√c. it never entered the running gel. d. substrates didn't enter the gel.

Part B - Short Answers. Answer these questions in one sentence or equivalent. 5 points each.

1. Cleavage of amino acid thiazolinones from the peptide chain uses anhydrous trifluoroacetic acid (TFA), while conversion of the thiazolinones to thiohydantoins uses TFA - H₂O 50/50 mixture. Why?

If aqueous TFA were used to cleave off thiazolinones, hydrolysis would occur at other points in the peptide chain, generating new, spurious amino termini.

2. Write a balanced chemical reaction for the reaction catalyzed by d-amino acid oxidase.

CH₃CH(NH₃⁺)COO^- + O₂ + H₂O Æ CH₃COCOO^- + H₂O₂ + NH₄⁺

3. EXTRA CREDIT: Write a balanced chemical reaction for the net reaction catalyzed by d-amino acid oxidase + catalase.

2CH₃CH(NH₃⁺)COO^- + O₂ Æ 2CH₃COCOO^- + 2NH₄⁺

Part C - Problems. Show your calculations, and identify the answer clearly.

1. Glutamic acid has three pK_as, approx. 2.0, 4.0, 9.8.
a. Write the structure of monosodium glutamate, with proper ionic form of the ionizable groups (1 pt).

Na^{+ -}OOCCH₂CH₂CH(NH₃⁺)COO^-

b. To one liter of 0.1 M monosodium glutamate is added 3.3 ml of 10 M NaOH. What is the pH? (Which pK_a will you use in the calculation?) (4 pts)

Use pK₃ = 9.8. One L of 0.1 M = 100 mmoles; 3.3 ml 10 M OH^- = 33 mmoles. Thus the amino group will be 1/3 titrated, [glu⁼] = 1/2[glu^-], pH = 9.8 + log (1/2) = 9.8 - log 2 = 9.8 - 0.3 = 9.5.

2. The following data are obtained from Coomassie Blue reaction with standard ovalbumin, 0.2 mg/ml (fill in blanks as necessary) (6 pts):

ml 0 0.025 0.05 0.1 0.2 0.3

mg

A₅₉₅ .366 .442 .525 .661 .870 1.08

A₄₆₆ .665 .583 .541 .475 .390 .352

.550 .76 ,97 1.39 2.23 3.07

and from reaction with a 1:25 dilution of a
stock solution of an unknown protein:

ml 0.05 0.15 0.3

A₅₉₅ .486 .688 .939

A₄₆₆ .549 .442 .366

,886 1.56 2.566

mg .008 .024 ,048

Calculate the concentration of the stock
solution. Use the graph at right if you
wish, or an appropriate equation.

The above all indicate the concentration of the dilute solution is 0.16 mg/ml, that of the stock solution 4.0 mg/ml.

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 0.91 6,500 carbonic anhydrase 0.523 29,000
a-lactalbumin 0.71 14,200 ovalbumin 0.408 45,000
trypsin inhibitor 0.619 20,000 albumin, bovine serum 0.308 66,000

An unknown protein has an R_m of 0.38 on the same gel. Calculate its molecular weight (use graph below, or fit the standard molecular weights to an appropriate equation).

Constructed from R_m = 3.2 - 0.6 log mol. wt.; thus 3.2 - R_m = 0.6 log mol. wt. of unknown = 4.7, mol. wt. = 50,118.

4. (4 pts) Twenty-five microliters of a 1:10 dilution of purified d-amino acid oxidase is added to 3.0 ml assay mixture in the polarograph. The initial rate of oxygen utilization is 0.144 chart widths/min (1.44 cm/min). If 3.0 ml assay mix contains 0.78 µmole O₂ (i.e. full scale = 0.78 µmole), calculate the activity in µmoles/min·ml enzyme of the stock enzyme.

0.144 x 0.78 x = 45 units/ml

5. a) (8 pts) The following rates of enzymatic oxidation of samples of 0.02 m dl-methionine are observed in the peroxidase assay (assay volume 3.0 ml):

ml dl-methionine 0.025 0.05 0.10 0.20 0.4 0.8 1.2

[d-methionine], mm: 0.0.83 0.167 0.33 0.67 1.33 2.67 4.0

∆A₅₀₀/min 0.094 0.148 0.210 0.264 0.303 0.328 0.337

Calculate the methionine concentrations, the K_m for this substrate, and V_max in mmoles/ min for this amount of enzyme (hint for speed: calculate in ∆A₅₀₀, then convert V_max to mmoles/min [e₅₀₀ of peroxidase product = 14,250]. Use graph below if desired.)

Constructed from V_max = 0.075 µmole/min = 0.35625 A/min, K_m = 0.233 mM.

(b)(1 pt) If the enzyme used was 0.1 ml of a 1:80 dilution, what is the V_max in mmoles/min^. stock enzyme?

0.075 µmole/min x = 60 u/ml

(c)(1 pt) If the stock enzyme had a protein concentration of 1.95 mg/ml, and the molecular weight (per subunit) is 39,000, what is the turnover number?

1.95 mg/ml ∏ 39,000 mg/mmole = 5 x 10^-5 mmole/ml = 5 x 10^-2 µmole/ml.
60 µmole/min·ml ∏ 5 x 10^-2 µmole/ml = 1200 min^-1