December 15, 1992 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.   At. 0.3 pH unit (log10 2) below the pKa of a buffer, the concentration of the basic form equals
a. the concentration of the acidic form.
b. one half the concentration of the acidic form.
c. one half the total concentration of the buffer.
d. two thirds of the total concentration.

2.   Change of the pH of a buffer as it is diluted occurs mainly when the buffering species include a
a. cation.                  b. unionized form.                  c. monoanion.                  d. dianion.

3.   Which method for protein determination has a high (and potentially variable) blank absorbance vs. H2O?
a. Lowry                  b. biuret                  c. Coomassie Blue                  d. ultraviolet abs.

4.   Which method for protein determination would be least appropriate for measurement of protein during purification of d-amino acid oxidase in presence of 0.1% Na benzoate?
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.  One ml of this first dilution is diluted with 4 ml water to yield the second dilution.  Three tenths of a milliliter of this second dilution is found to contain 0.012 mg protein.  What is the protein concentration of the stock solution?
a. 100 mg/ml                  b. 50 mg/ml                  c. 10 mg/ml                  d. 8 mg/ml

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

7.   In which protein determination method would polyglycine give a higher absorbance per mg than polytryptophan?
a. Lowry                  b. biuret                  c. Coomassie Blue                  d. ultraviolet abs.

8.   Treatment of the protein with phenyl isothiocyanate (PITC) after dimethylaminoazoben­zene isothiocyanate (DABITC) is to
a. insure that the a-amino terminus is fully modified, and will be fully cleaved.
b. insure that lysine e-amino groups are fully modified.
c. cleave off the terminal DABTH-amino acid.
d. facilitate the removal of excess DABITC.

9.   What does extraction of the DABITC reaction mixture with heptane-ethyl acetate remove?
a. excess DABITC                  b. excess PITC                  c. pyridine                  d. all of these

10. From the fact that DABTH-aspartic acid is first off the hplc column and DABTH-leucine is last off, you should reason that the DABTH-amino acids stick to the column by
a. hydrophobic interactions                  b. ionic interactions.
c. hydrogen bonds.                          d. p-p interactions.

11. One of the following amino acids definitely is not cleaved off by carboxypeptidase A:
a. histidine                  b. leucine                  c. asparagine                  d. proline

12. Which of these amino acids should have the lowest mobility in the chromatographic system we used?
a. serine                  b. leucine                  c. asparagine                  d. proline

13. Which of the following is not truly an advantage of (NH4)2SO4 in protein precipitation?
a. neutral reaction (solution pH = 7)                  b. high solubility
c. high ionic strength                  d. lack of direct interaction with proteins

14. Which purification procedure is likely to achieve greatest purification as a single step?
a. (NH4)2SO4 precipitation                  b. heating                  c. gel filtration                  d. hydroxylapatite

15. In gel filtration, the first proteins to elute are
a. the largest                  b. the smallest                  c. the most charged                  d. the least charged

16. Specific activity is
a. mmoles/min.ml enz.  b. mmoles/min.mg protein. c. mmoles/min.ml assay. d. total units.

17. Benzoate is added to solutions for purification of d-amino acid oxidase because it
a. protects it from air oxidation.                  b. protects it from protease action.       
c. stabilizes FAD on the enzyme.                  d. all of the above.

18. In assaying enzyme activity during purification, we keep all but one of these constant:
a. substrate concentration                  b. enzyme concentration                  c. pH                  d. temperature

19. What is not an inherent advantage of a stop-time assay?
a. Many samples or conditions can be run at the same time.
b. It can be made more sensitive by increasing the time of incubation.
c. It shows directly whether the initial rate is being observed.
d. It allows separation of product and starting material, when they are not distinguished by the property measured (e.g. radioactivity).

20. To get activity of the stock solution in the peroxidase assay in mmoles/min.ml enzyme, multiply the ∆A500 by
a. x                   b. x
c. x x                   d. x dilution factor

21. The Km is
a. Vmax/2                  b. not affected by presence of a competitive inhibitor
c. that [S] at which v = Vmax/2                  d. affected by amount of enzyme used

22. Determination of the Km for O2 by a single run of the polarograph requires that
a. the instrument measure O2                  b. the assay be continuous
c. Km be not much less than initial [O2]                  d. all of the above

23. What property of a protein is measured in SDS polyacrylamide gel electrophoresis?
a. size of the native enzyme                  b. size of denatured subunits
c. biological activity                          d. charge density of the native protein

24. The key property of the stacking gel is
a. pH several units lower than pKa of glycine.                  b. low (4%) acrylamide concentration.              
c. presence of a zwitterionic buffer.                  d. all of the above.

25. 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.

26. Most of the RNA in leaf tissue is
a. messenger RNA                  b. nuclear RNA                  c. transfer RNA                  d. ribosomal RNA

27. Basic hydrolysis of RNA depends on presence of a free
a. 2'-OH                  b. 3'-OH                  c. 5'-OH                  d. base

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

29. Our elution of the nucleotides from DEAE-Sephadex uses a
a. stepwise gradient                  b. linear gradient                  c. logarithmic gradient                  d. concave gradient

30. Observation that the ratio of mmoles by orcinol to mmoles by UV absorption is lower for CMP and UMP than for AMP and GMP suggests that
a. the glycosidic bond to purines hydrolyzes more easily than that to pyrimidines.
b. orcinol reacts only with purines.
c. the 2' (or 3') phosphate does not affect the reaction.
d. the orcinol product with CMP and UMP does not absorb at 660 nm.

 

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

1.   (8 points) The following A595 values were recorded for samples of a 1:100 dilution of bovine serum albumin, 10.0 mg/ml, in the Coomassie Blue protein determination:

      ml BSA                  0.05                  0.10                  0.15                  0.20                  0.25                  0.30           
A595                  0.130                  0.240                  0.365                  0.495                  0.630                  0.780

      The following A595 values are recorded for samples of a 1:50 dilution of a protein solu­tion of unknown concentration:

      ml protein                  0.05                  0.15                  0.30
A595                  0.077                  0.178                  0.337

      Calculate (1) the concentration of the diluted solution (6 pts); (2) the concentration of the stock solution (2 pts).  Use graph below if desired.  Show answers clearly.

 

 

 

2a. (6 points) If the pK2 of alanine is 9.87, pK3 9.0, what is the concentration of the anion of d-alanine in a solution of total dl-alanine concentration 0.06 m at pH 9.50?

b.   (4 pts) What will be the total dl-alanine concentration in a solution containing the same concentration of d-alanine anion as calculated in part a, but at pH 8.3?  (Hint to get you started: the total has to be larger.)

3. The millimolar extinction coefficients (units: ml/mmole.cm) for GMP and UMP are:

                        e260                                    e280                                    e260/e280

      GMP                  11.7                  8.0                  1.46

      UMP             9.8                  3.5                  2.8

      A solution containing GMP and UMP has A260 = 0.879, A280 = 0.505.  Calculate the molar concentration of the two nucleotides present. (10 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          0.163          0.333          0.500          0.667        0.833

      Calculate the slope (conversion factor to convert observed A560 to mmoles pyruvate.  Not the molar extinction coefficient.) (2 pts)

      (b) The following A560 values were observed from the enzymatic oxidation of samples of 0.025 m d-valine, diluted to an assay volume of 1.0 ml (3 ml in the spectrophotometer):

      ml d-valine          0.025            0.05            0.10            0.20            0.40            0.60          0.80

      [d-valine], mm:

      A560     0.091          0.158          0.250          0.353          0.444          0.486        0.511

      Calculate Km for this substrate, and Vmax in mmoles/min for this amount of enzyme (hint for speed: calculate in A560, then convert Vmax to mmoles/min.  Use graph below if desired.) (8 pts)

 

 

(c)(1 pt)  If the enzyme used was 0.1 ml of a 1:200 dilution, what is the Vmax in mmoles/min. stock enzyme?

(d)(1 pt) If the stock enzyme had a protein concentration of 3.6 mg/ml, and the molecular weight (per subunit) is 50,000, what is the turnover number?