December 20, 1996 name______________________section___

Final Examination - 115:413 Experimental Biochemistry

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

1. You are titrating a solution, with an indicator present. The indicator changes color slowly as base is added. This indicates that
a. the end point of the titration has been completed.
b. the solution is buffered, and the pK_as of the buffer and the indicator are similar.
c. the indicator has two pK_as.
d. the solution is not buffered.

2. Histidine has three pK_as, which for the free amino acid are 1.9 (the COOH), 6.0 (the imidazole ring), 9.4 (the a-amino group). In solution at pH 6.0, the average charge on the histidine molecules present is
a. -1 b. 0 c. +0.5 d. +1

3. Which method for protein determination gives most constant results from protein to protein. irrespective of amino acid composition?
a. biuret b. Lowry c. Coomassie Blue d. ultraviolet

4. A lysozyme solution, 1.0 mg/ml, gives a higher absorbance at 280 nm than a 1.0 mg/ml solution of ovalbumin because
a. its molecular weight (14,000) being smaller than that of ovalbumin, the solution contains more moles of protein.
b. its peptide bond content per mg protein is higher.
c. it contains more arginine per molecule than ovalbumin.
d. it contains more tryptophan per molecule than ovalbumin.

5. A plot of A₂₈₀ vs. mg ovalbumin falls below the straight line expected by linear extrapolation at A₂₈₀ > 1.5. This is probably because
a. Beer's law is not valid at high protein concentration.
b. reaction with the protein is incomplete, due to exhaustion of the reagent.
c. the protein yields a cloudy precipitate at this concentration.
d. the spectrophotometer is detecting scattered light of other wave lengths, significant when so little specific light reaches the detector.

6. The absorbance at 595 nm observed in the Coomassie Blue method is due to
a. an adsorbed blue form of the dye. b. the Cu⁺⁺ complex with peptide bonds.
c. reduced phosphomolybdate. d. reduction of Cu⁺⁺ to Cu⁺.

7. In the Lowry method, reagent B should be
a. added dropwise to prevent overheating.
b. added slowly and carefully, lest the protein be denatured.
c. added and mixed quickly, since the half-life in alkaline solition is short.
d. added immediately after the copper reagent.

8. The reaction of proteins with p-dimethylaminoazobenzene (DABITC) is carried out in sealed vials under a nitrogen atmosphere because
a. oxidation of proteins by atmospheric oxygen makes them unreactive.
b. replacement of sulfur by oxygen in the thiocambamoyl product makes it uncleavable.
c. phenylisothiocyanate is volatile. d. pyridine is smelly.

9. The addition of phenyl isothiocyanate after 50 minutes of reaction is to
a. ensure that the amino-terminal residue is fully reacted and thus cleaved off.
b. ensure that lysine e-amino groups are fully reacted.
c. react with excess DABITC.
d. push the reaction further by mass action.

10. Real spots of amino acid DAB-thiohydantoins may be best distinguished from other spots (internal standard, products of DABITC reaction with PITC and breakdown) by
a. being beyond the diagonal line through DABITC and the internal standard
b. their R_f values. c. being pink, rather than violet. d. being invisible.

11. A disadvantage of carboxypeptidase digestion compared to chemical cleavage through a thiohydantoin is that
a. it is not well adapted to use in a lab class.
b. no carboxypeptidase cleaves off all amino acids.
c. it may remove several amino acids in quick succession.
d. it is an enzymatic reaction.

12. To be separated appreciably in thin layer chromatography, and characterized by their R_f, two components must be
a. appreciably soluble in the nonpolar phase.
b. appreciably soluble in H₂O.
c. appreciably soluble in the nonpolar phase and H₂O.
d. appreciably soluble in neither H₂O nor the nonpolar phase.

13. One of the following generally does not denature enzymes:
a. extreme pH b. heating c. foaming d. high salt conc.

14. The purpose of benzoate in solutions used for purification of d-amino acid oxidase is
a. to stabilize the protein structure. b. to stabilize the FAD on the protein.
c. to buffer the solution. d. to inhibit the growth of bacteria.

15. In judging whether an inhibitor of an enzyme has been removed during purification, the best criterion is an increase, at a later purification step, in
a. units/ml. b. total units. c. specific activity. d. purification factor.

16. The K_m of an enzyme is defined as
a. the slope of a Lineweaver-Burk plot b. the x intercept of a Lineweaver-Burk plot
c. V_max/2 d. [S] when v = V_max/2

17. Which of the following terms describes the peroxidase assay only, in comparison to other assays we used?
a. coupled b. spectrophotometric c. continuous d. product appearance

18. Hydroxylapatite chromatography is a particularly effective means of purifying d-amino acid oxidase because
a. you can see the enzyme on the column
b. the enzyme is eluted from the column by a particularly low phosphate concentration
c. the column is eluted using a gradient of phosphate concentration
d. the column can be run at room temperature

19. In the comparison of the three assays of d-amino acid oxidase, the peroxidase and polarograph assays generally showed less activity (units/ml stock enzyme) than the pyruvate assay. One of the following is not a reasonable explanation for this:
a. the peroxidase assay is run at room temperature, the pyruvate assay at 37°.
b. benzoate in the enzyme is not diluted out enough in the polarograph and inhibits.
c. the peroxidase and polarograph assays were run a week later than the pyruvate assay.
d. the pyruvate assay is run for 10 minutes, therefore is inherently more sensitive.

20. The polarograph assay is useful for determination of the K_m of d-amino acid oxidase for oxygen because
a. it shows the rate of the reaction continuously.
b. it shows the oxygen concentration continuously.
c. it shows the rate of the reaction at a variety of oxygen concentrations.
d. all of the above.

21. In determination of the K_m of d-amino acid oxidase for its substrate, 0.05 ml stock 0.06 M dl-alanine - 0.1 M PP_i is used, with 0.45 ml 0.1 M PP_i, 0.1 ml 0.1 mM FAD, 0.1 ml enzyme, 0.3 ml H₂O. The reaction is stopped after 10 min with 0.5 ml 2,4-dinitrophenylhydrazine, and 1.5 ml 2M NaOH is added to make the solution basic. The substrate concentration which should be used in calculation of the K_m is
a. 1.5 mM b. 3 mM c. 0.5 mM d. 0.05 M.

22. In centrifugation, doubling the speed (revolutions per minute) of the centrifuge, using the same sized centrifuge head, increases the centrifugal force by a factor of
a. two. b. four. c. six. d. eight.

23. What is the function of ammonium persulfate in polyacrylamide gel electrophoresis?
a. It denatures proteins so that shape is not a factor in separation.
b. It associates with proteins so that they have a constant charge density.
c. It generates free radicals which initiate acrylamide polymerization.
d. It buffers the gel.

24. We used Tricine in the reservoir (electrode) buffers rather than glycine. What is the most important characteristic of this compound in gel electrophoresis as we did it?
a. Very little of it is an anion at the pH of the stacking gel.
b. Very little of it is an anion at the pH of the running gel.
c. It is a buffer with pK_a = 8.15.
d. It associates with proteins so that they have a constant charge density.

25. What is an important reason for also electrophoresing a purified protein by a 'native' procedure for determining molecular weight, such as several gels of different % acrylamide or a gel with a gradient of acrylamide concentration?
a. To show whether isozymes, of identical mol. weight but different charge, are present.
b. To show whether the mol. weight observed in SDS electrophoresis has been influenced by the shape of the protein.
c. To show whether the native protein is made up of subunits.
d. To be able to observe the activity of the enzyme in the gel.

26. The product of basic hydrolysis of RNA is
a. nucleoside 5'-phosphates. b. nucleoside 3'-phosphates.
c. a mixture of nucleoside 2'- and 3'phosphates. d. free nucleosides.

27. Chromatographic separation of the ribonucleotides depends on differences in
a. pK_as of the phosphates. b. pK_as of the bases.
c. affinity of the phosphates for DEAE-Sephadex d. hydrophobicity of the bases.

28. The nucleotide with highest A₂₆₀/A₂₈₀ ratio is
a. AMP b. CMP c. GMP d. UMP

29. The material precipitated by adding HClO₄ to the RNA hydrolyzate is
a. unhydrolyzed RNA b. carbohydrate c. protein d. KClO₄

30. The orcinol reaction measures
a. RNA b. aromatic bases. c. phosphate d. ribose

Part B - Short answers.

Answer these questions in one sentence (longer answers will not be read.) 5 points each.

1. An amino-terminal lysine on a polypeptide chain will yield three colored spots in the DABITC Edman degradation method, blue, violet and pink. Explain what they are.

2. The molar extinction coefficient of pyruvate dinitrophenylhydrazone is about 9600 L· mole^-1cm^-1 at 560 nm, 12,000 L·mole^-1cm^-1 at 416 nm. The extinction coefficient of the peroxidase product is 14,250 L·mole^-1cm^-1 at 500 nm. (The polarograph assay does not have an extinction coefficient, since it is not spectrophotometric, but a similar calculation, 1 chart width/0.26 mmole·ml x 10³ mmole/mmole, yields a value of 3846 L/mole.) Yet the pyruvate assay is considered the most sensitive of the three assays. Why?

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

1. (6 pts) The following amounts of standard ovalbumin solution, 0.4 mg/ml, and of a 1:10 dilution of an unknown solution gave the following A₇₄₀ in a Lowry protein assay:

ml std. ovalbumin 0 0.05 0.10 0.15 0.20 0.25 ml unknown 0.05 0.15 0.25

A₇₄₀ 0 .122 .246 .364 .490 .610 0.109 .336 .552

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

2. The molecular weight of the buffering compound Tricine, (HOCH₂)₃CNHCH₂COOH, is 182, and the pK_a is 8.15.
a. (1 pt) Which would you add to a solution containing only Tricine to prepare a pH 8.0 buffer, HCl or KOH?

b. (6 pts) How much Tricine (grams) and 5M HCl or KOH (ml) would you use to prepare 1 liter of 0.1 M Tricine buffer pH 8.0?

3. (6 pts) The millimolar extinction coefficients of UMP and GMP at 260 and 280 nm are:

e₂₆₀ e₂₈₀

UMP 9.8 2.8 A solution has A₂₆₀ = 1.01, A₂₈₀ = 0.500. What are the concen-

GMP 11.7 8.0 trations (mM) of UMP and GMP in it?

4. The following A₅₆₀ values were observed for pyruvate formation from various dl-alanine concentrations by 0.1 ml of a 1:400 dilution of final enzyme in our standard assay (volume 1.0 ml, 10 minutes at 37°, then add 0.5 ml DNPH and 1.5 ml 2M NaOH) (molar extinction coefficient = 10,000 L·mole^-1cm^-1):

ml 0.06 M dl-ala 0.020 0.040 0.060 0.010 0.020 0.030 0.050

A₅₆₀ 0.265 0.486 0.628 0.820 1.06 1.17 1.25

a. (4 pts) Calculate either [S]/v (Woolf plot) or 1/[S] and 1/v (Lineweaver-Burk plot) and plot on the graph below. Use any form of v you wish (A₅₆₀, mmoles, mmole/min, mmole/ min·ml dilute enzyme, mmole/min·ml stock), but label the axes clearly.

1/[S]

1/v

[S]/v

b. (4 pts) Determine K_m (mM) and V_max (in whatever units you used for the plot).

c. (1 pt) Calculate V_max as mmole/min·ml stock enzyme.

d. (2 pts) If the protein concentration of the final enzyme was 3.08 mg/ml, what is the specific activity? What is the turnover number? (Mol. wt. 50,000 daltons)