December 18, 1995 name______________________section___

Final Examination - 115:413 Experimental Biochemistry - Master

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

1. You have a solution, approximately 1 mM, of the coenzyme NADH (e₃₄₀ = 6220 L/mole·cm). You wish to determine its concentration spectrophotometrically at 340 nm, but you don't want to use too much of it. How much of this solution should you dilute to 1 ml to achieve an absorbance approximately = 0.2?
a. 3 µl b. 10 µl ÷c. 30 µl d. 300 µl

2. Fifty microliters of a solution are diluted with 2.45 ml water; 0.3 ml of this solution is diluted with 2.7 ml water. The overall dilution is
a. 1:150 b. 1:441 ÷c. 1:500 d. 1:1500

3. One ml of 1M KOH is added to 100 ml of 5 mM Tris Cl buffer, pH 8.3 (the pK_a is 8.3). What will the new pH be, approximately?
a. 8.4 b. 8.6 c. 9.8 ÷d. 11.7

4. Recording 'room temperature' would be important in preparing which buffer solution? ÷a. Tris/TrisHCl b. Na acetate c. K phosphate d. Na citrate

5. A low net absorbance reading is most likely to be in error in which method?
a. biuret b. Lowry ÷c. Coomassie Blue d. UV

6. 5% transmittance corresponds to what absorbance?
a. 0.05 b. 0.3 c. 1.0 ÷d. 1.3

7. In which method for protein determination is the reagent in acid?
a. biuret b. Lowry ÷c. Coomassie Blue d. UV

8. Which protein determination method is most subject to interference by other compounds?
a. biuret ÷b. Lowry c. Coomassie Blue d. UV

9. In the Edman degradation procedure we used, phenyl isothiocyanate is added
÷a. to ensure that all of the N-terminal amino acid is removed at the cleavage step.
b. to couple with the DAB-thioureido amino acid to make it colored.
c. to block e-amino groups on lysine residues.
d. to react with excess DABITC.

10. We used carboxypeptidase A, which cleaves uncharged and acidic amino acids from proteins. Other carboxypeptidases are available which cleave off almost all amino acids. However, even the most general of these cannot release terminal
a. arginine b. lysine c. cysteine ÷d. cystine

11. In our chromogenic Edman degradation method, one N-terminal amino acid will yield three colored spots. It is
a. arginine ÷b. lysine c. cysteine d. glutamine

12. Di-substituted lysine (a-DAB-e-DABthiocarbamoyl-lysine) comes off the hplc column even later than DAB-leucine, presumably due to
a. ionic interactions with the e-amino group.
b. polar interactions with the thioureido group.
c. additional hydrogen bonding.
÷d. hydrophobic interaction with the added DAB group.

13. If K', the functional distribution coefficient of solutes between moving and stationary phases, = , then R_f is related to it by
a. R_f = 1/K' b. R_f = 1+K'/K' ÷c. R_f = K'/1+K' d. R_f = K'/1-K'

14. In the purification of d-amino acid oxidase, if at the acid supernatant step you have 350 ml of enzyme preparation, activity 5.0 units/ml, what is the maximum likely activity in the redissolved (NH₄)₂SO₄ precipitate (volume 50 ml)?
a. 15 units/ml b. 25 units/ml ÷c. 35 units/ml d. 50 units/ml

15. Specific activity is defined as
a. units/ml b. units at step_n/units at step_n-1
÷c. units/mg protein d. units/mg at step_n/units/mg at step_n-1

16. Why is the (NH₄)₂SO₄-precipitated enzyme preparation dialyzed vs. 0.1% Na benzoate?
÷a. To keep the coenzyme FAD on the enzyme. b. To inhibit it during dialysis.
c. To inhibit the action of other enzymes. d. To keep the ionic strength up.

17. d-amino acid oxidase is eluted from hydroxylapatite by competing
÷a. phosphate ions b. crotonate ions c. Ca⁺⁺ ions d. benzoate ions

18. In the pyruvate assay of d-amino acid oxidase activity, a drop-off from linearity at A₅₆₀ >1.0 (A₅₆₀ less than expected for the amount of enzyme used) is most probably because
÷a. O₂ has been used up faster than it diffuses in from the atmosphere.
b. the substrate d-alanine has been depleted before the end of the 10 min incubation.
c. the enzyme is less efficient when at high concentration.
d. Beer's law does not hold at A₅₆₀ >1.0.

19. In the polarograph assay of d-amino acid oxidase, how do you expect the presence of catalase to affect the observed activity (decrease of O₂ concentration) if the catalase is fully active?
a. No d-amino acid oxidase activity will be observable.
÷b. The activity will be 1/2 what it is in absence of catalase.
c. The activity will be the same as in absence of catalase.
d. The activity will be greater than in absence of catalase.

20. Which of the assay methods you used is a coupled assay?
a. The Lowry assay. b. The pyruvate assay.
c. The polarograph assay. ÷d. The peroxidase assay.

21. You calculate your value of e₄₆₀ of d-amino acid oxidase to be 16,000 L/mole·cm, greater than that reported by Yagi. Which of the following is the most reasonable explanation for the discrepancy?
a. Your protein preparation is impure, containing other proteins which do not absorb at 460 nm.
÷b. d-amino acid oxidase produces less color per mg protein in your protein determination method than bovine serum albumin.
c. The spectrophotometer gave too high a reading for A₄₆₀.
d. Your enzyme is partially depleted of FAD.

22. For your first assay in determination of the K_m for d-alanine, you use 0.025 ml 0.06 M dl-alanine - 0.1 M PP_i, 0.575 ml 0.1 M PP_i, 0.1 ml FAD - catalase, 0.1 ml enzyme (1:400 dilution of stock), and 0.2 ml H₂O, followed by 0.5 ml 2,4-dinitrophenylhydrazone and 1.5 ml 2M NaOH to develop the color. What is the concentration of d-alanine in this assay, that you should use in calculation of the K_m?
÷a. 0.75 mM b. 1.25 mM c. 1.5 mM d. 25 mM

23. In gel filtration chromatography of proteins
a. the smallest protein molecules emerge from the column first.
÷b. the largest protein molecules emerge from the column first.
c. the salt molecules emerge from the column first.
d. the proteins are eluted by competition of salt ions for charges on the column.

24. In the gel electrophoresis system we used, the principal function of the stacking gel is
a. to separate the proteins by size. b. to separate the proteins by charge density.
c. to renature the proteins. ÷d. to concentrate the proteins.

25. The most important feature of the upper reservoir buffer is
a. that it keeps the proteins denatured.
÷b. that the anion has low net mobility at the pH of the stacking gel.
c. that the anion has low net mobility at the pH of the running gel.
d. that it buffers well.

26. The most hazardous chemical used in the RNA preparation procedure is
a. chloroform. b. isoamyl alcohol. ÷c. phenol. d. perchloric acid.

27. The 70% ethanol step in RNA purification
a. redissolves the RNA. b. separates remaining proteins.
c. separates DNA from RNA. ÷d. removes residual phenol and salts.

28. The molecular weight of an RNA molecules 3000 nucleotides long is
a. 10⁴. b. 10⁵. ÷c. 10⁶. d. 10⁷.

29. The structure of the charged side chain of DEAE-Sephadex is
a. -CH₂CH₂NH⁺(CH₂CH₂OH)₂ b. -CH₂CH₂N⁺(CH₂CH₃)₂(CHOHCH₃)
c. -CH₂CH₂NH⁺CH₂CH₂NH⁺(CH₂CH₃)₂ ÷d. -CH₂CH₂NH⁺(CH₂CH₃)₂

30. To generate a convex gradient, the bottle in which mixing occurs should
a. have a larger cross-section than the other bottle.
÷b. have a smaller cross section than the other bottle.
c. be taller than the other bottle.
d. be shaped like an Erlenmeyer flask.

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

1. A ribose standard solution, 0.15 mM, gives the following results in the orcinol method:

ml ribose 0 0.3 0.6 0.9 1.2 1.5
µmoles ribose
A₆₆₀ 0 0.144 0.289 0.465 0.573 0.722

Samples of an RNA solution give the following results in the orcinol method:

µl RNA 0 10 30 50
A₆₆₀ 0 0.135 0.402 0.672

a. (5 points) What is the concentration (mM ribose) of the stock RNA solution?

Slope of standard curve = 0.48 A/ml = 3.2 A/µmole. Slope of A vs. ml RNA solution = 13.44 A/ml. Slope of A vs. ml RNA/slope of standard curve = 13.44/3.2 = 4.2 µmole/ml = 4.2 mM.

b. (1 point) If the average molecular weight of a nucleotide in RNA is 320, what would the corresponding concentration of the stock RNA be in mg/ml?

4.2 µmoles/ml x 0.32 mg/µmole = 1.344 mg/ml

c. (3 points) Twenty microliters of the stock RNA, diluted to 2.0 ml, has an A₂₆₀ = 0.650 (1 cm path length cell). Using E₂₆₀ = 25 ml/mg·cm, what RNA concentration does this correspond to?

0.65 A/25 = 0.026 mg/ml dilute solution; x 100 = 2.6 mg/ml

d. (3 points) Explain in words why the two values do not agree.

For ribose molecules to react in the orcinol assay, the glycosidic bond to the nucleotide base must hydrolyze. This happens only for the purine nucleotides, about half the total.

2. The initial pH of an acid solution (volume 50 ml) is 2.17. It is titrated with 0.5 M KOH, with the following results:

ml KOH added: 1.0 2.0 4.0 6.0 8.0 9.0 10.0
pH 2.40 2.75 3.17 3.53 3.95 4.30 10.0
(i.e. the titration is just complete at 10.0 ml; no water correction needed)

a. (5 pts) What is the pK_a of the acid ? You shouldn't need the graph below, but I give it in case you do.

Since the acid solution is fully titrated at 10.0 ml, it is half titrated at 5.0 ml, half way between 4.0 and 6.0 ml. Halfway between the corresponding pH values, 3.17 and 3.53, is 3.35.

b. (1 pt) What is the concentration of the acid? Since 10 ml of 0.5 M KOH = 5 mmoles fully titrates it, the solution contains 5 mmoles acid in 50 ml, it is 0.1 M.

c. (4 pts) What is the pH when 7.0 ml 0.5 M KOH have been added?

When 7.0 ml base has been added, the solution is 0.07 M in basic form of the acid, 0.03 M in remaining acid. pH = pK_a + log = 3.35 + log 2.33 = 3.35 + 0.37 = 3.72.

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

R_m = m log mol. wt. + b. m = -0.489, b = 2.673. R_m = 0.38 corresponds to log mol. wt. = 4.69, mol. wt. = 49,100.

4. 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 0.256 chart widths/min (2.56 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.

0.256 chart widths/min x 0.78 µmole/chart width = 0.20 µmoles/min; x 10/ 0.025 ml = 80 units/ml.

5. a) (8 pts) The following rates of enzymatic oxidation of samples of 0.030 m dl-norleucine are observed in the peroxidase assay (assay volume 3.0 ml):

ml dl-norleucine 0.025 0.05 0.10 0.20 0.4 0.8 1.2

[d-norleucine], mm: 0.125 0.25 0.5 1.0 2.0 4.0 6.0

∆A₅₀₀/min 0.046 0.081 0.132 0.193 0.250 0.294 0.312

Calculate the norleucine 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.)

The rates were set up from V_max = 0.35625 A/min = 0.075 µmole/min, K_m = 0.85 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^.ml stock enzyme?

0.075 µmole/min x 80/0.1 = 60 µmole/min^.ml stock

(c)(1 pt) If the stock enzyme had a protein concentration of 1.2 mg/ml, and the molecular weight (per subunit) is 50,000, what is the turnover number?

60 µmole/min·ml ÷ 1.2 mg/ml = 50 µmol/min·mg; ÷ 50 mg enzyme/µmol = 1 min^-1 = 60 sec^-1.