December 16, 1998                                                                                                               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 glass pipets?
a. Checking the top end for a ground glass ring.
b. Expelling solution slowly from a Mohr pipet (non-blow-out).
c. Grasping the pipet in the middle when inserting into a PiPump.
d. Holding the pipet, as well as the PiPump, when moving to the tube into which you will pipet.

2.     0.1 ml of stock solution is diluted with 2.4 ml H2O.  Fifty microliters of this solution is diluted with 0.95 ml H2O.  The overall dilution is
a. 1:240                                         b. 1:250                                         c. 1:400                                         d. 1:500

3.     The pKa of a weak acid or base is most easily determined as
a. the pH halfway between the two pIs.
b. the pH where the compound is half titrated.
c. the pH of maximum slope of a plot of ml acid or base vs. pH.
d. the pH of maximum slope of a plot of pH vs. ml acid or base.

4.     If the pKas  of phthalic acid are 3.0 and 5.0, the pKa of ammonia (NH4+ Ć NH3 + H+) is 9.3, what will the pH of an 0.1 M ammonium biphthalate solution be (approximately)?
a. 4.0                                             b. 5.0                                             c. 7.15                                           d. 9.3

5.     The units of an extinction coefficient e are:
a. L·g-1cm-1                                  b. L·mole-1cm-1                            c. ml·mg-1cm-1                              d. L·mole-1cm

6.     Which protein reagent can be used to measure concentration of any protein equally well, irrespective of its amino acid composition?
a. biuret                                       b. Lowry                                       c. Coomassie Blue                     d. A280

7.     Why is the new Cary spectrophotometer particularly useful for the Zor & Seliger A595/A466 Coomassie Blue method?
a. it reads absorbance up to 10.
b. it prints out the results.
c. it measures A595, A466 and their ratio, all at once.
d. it requires less sample volume than a Spectronic 88.

8.     Which of the following amino acids does not react in the Lowry method?
a. tyrosine                                   b. cysteine                                   c. arginine                                   d. tryptophan.

9.     In protein purification, dialysis is used most generally
a. to remove inhibitors from the protein solution.
b. to precipitate out proteins which are insoluble at low salt concentration.
c. to decrease the volume of the protein solution.
d. to lower concentration of low mol. wt. compounds in the protein solution.

10. A unit of enzyme activity is usually
a. one millimole product produced/min (under specified conditions).
b. one micromole product produced/min (under specified conditions).
c. one micromole product produced per mg protein (under specified conditions).
d. one micromole of enzyme.

11. The three independently measured quantities in a protein purification table are
a. volume, units/ml, mg protein/ml.                                               b. units/ml, mg protein/ml, yield.
c. units/ml, mg protein/ml, spec. activity.                                     d. units/ml, total units, specific activity.

12. In the hydroxylapatite chromatography, the function of crotonate is
a. to buffer the solution.           b. to absorb at 280 nm.
c. to elute the enzyme.              d. to keep FAD on the protein.

13. The phenyl-Sepharose step is an example of
a. gel filtration                                                                                  b. hydrophobic chromatography
c. ion exchange chromatography                                                  d. affinity chromatography

14. Hydroxylapatite is a form of
a. calcium phosphate                b. phosphocellulose                  c. polyacrylamide                       d. polystyrene

15. In the peroxidase assay, the observed rate of reaction is often seen to increase with time.  This is probably because
a. benzoate is being released slowly from d-amino acid oxidase.
b. the d-amino acid oxidase is renaturing slowly during the assay.
c. it takes time to reach a steady-state peroxide concentration at which peroxidase is acting just as fast as d-amino acid oxidase.
d. it always takes time for an enzyme to reach its maximum velocity.

16. The chart trace in the polarograph assay is curved because
a. the enzyme is denaturing during the reaction.
b. accumulating peroxide is inhibiting the reaction.
c. substrate O2 is being used up.
d. the polarograph is an unreliable instrument.

17. The function of the stacking gel in polyacrylamide gel electrophoresis is
a. to denature proteins in the sample.                                           b. to separate proteins in the sample.
c. to protect the running gel from air.                                           d. to concentrate proteins in the sample.

18.  One of the following is not a property of acrylamide:
a. it is hydrophilic.                                                                           b. it polymerizes to form long chains.
c. it is toxic.                                                                                       d. it cross-links during polymerization.

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

20. Quantitation of protein bands in the Coomassie Blue-stained gel depends on what assumption?
a. All proteins present stain equally well with Coomassie Blue.
b. All proteins present are negatively charged.
c. All proteins present were denatured by SDS.
d. Staining is proportional to molecular weight..

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

22. Determination of purity of d-amino acid oxidase by isoelectric focusing has what advantage over using SDS gel electrophoresis?
a. You can determine the molecular weight of the native protein directly.
b. You can determine by assay of the same gel which band is d-amino acid oxidase.
c. Mobility is not affected by shape.
d. You do not need to stain with Coomassie Blue.

23. The biggest problem in preparation of good quality RNA from plants is
a. proteases.                               b. polyphenols.                           c. removal of phenol.                 d. ribonucleases.

24. Nucleic acids are commonly precipitated (in presence of cations) by
a. phenol.                                    b. chloroform.                              c. alcohols.                                  d. perchloric acid.

25. The net charge on nucleotide molecules at pH 5 is approximately
a. 0                                          b. -1                                        c. -2                                         d. varies with the nucleotide

26. The orcinol reaction measures the concentration of
a. ribose.                                      b. phosphate.                              c. nucleotides.                            d. RNA.

Part B - Short Answers.

1.  (3 pts)  Write the balanced chemical reaction for the reaction catalyzed by d-amino acid oxidase.

2.  (5 pts)  Three sets of pyruvate assay tubes for d-amino acid oxidase were set up, and after adding the same amount of enzyme to all tubes were incubated for various times at 37°.  Series A were incubated with no shaking; series B with constant gentle shaking; and series C without shaking, but contained catalase (added with the FAD).  The following A560 results were obtained:

        time                   0                  2'                 4'                 6'                10'               15'               20'
A560: A 0         0.20             0.39             0.58             0.97             1.20             1.40
              B           0                0.20             0.40             0.60             1.00             1.50             1.96
              C           0                0.20             0.40             0.60             0.99             1.45             1.88

Explain, in not more than three sentences total, why pyruvate production slows down after 10 min in series A, and why it does not in the other two series.  Assume that the spectro­photometer is accurate.

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

1. Succinic acid has two pKas, 4.1, 5.44.  How much 0.5 m KOH will it take to titrate 50 ml 0.1 M succinic acid to pH 5.2? (5 pts).

2.   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 0.109 chart widths/min (1.09 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 stock enzyme (5 pts).

3.  Do either a or b (6 pts).
a.  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

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

ml                          0.05         0.15          0.3

A595                       .490         .695         .917

A466                       .567         .465         .376

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

        b. 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                        0.114                                .343         .572               

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

 

4.  (5 points) Observed Rms and molecular weights of the standard proteins for molecular weight determination by SDS gel electrophoresis are as follows:

Protein                                       Rm                       mol. wt.              Protein                                                   Rm                   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 Rm of 0.275 on the same gel.  Calculate its molecular weight (use graph below, or fit the standard molecular weights to an appropriate equation).

 

 

5a. (6 pts)  The millimolar extinction coefficients (units: ml/mmole.cm)of UMP and GMP are:

                              e260              e280                  e260/e280     

        UMP            9.8               3.5                 2.8        A solution has A260 = 1.094, A280 = 0.620.  What are the

        GMP             11.7             8.0                1.46       concentrations (mM) of UMP and GMP in it?

 

        b. (3 pts)  1.0 ml of this solution gives an A660 = 0.522 in the orcinol reaction.  Using a slope of 9.0 A/µmole for the ribose standard curve, calculate the µmoles ribose observed.  Why does this not agree with the sum of µmoles UMP and GMP calculated above?

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

        ml dl-isoleucine                      0.075                0.15                 0.30               0.60                   0.90                  1.2                   1.5

        [d-isoleucine], mm:

        1/[d-ile], mm-1:

        ∆A500/min                                  0.135               0.220               0.321               0.417               0.463               0.490               0.507

        1/v or [d-ile]/v:

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

 

 

 

 

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

(c)(1 pt) If the stock enzyme had a protein concentration of 2 mg/ml, and the molecular weight (per subunit) is 39,000, what is the turnover number?