Bil 255 Spring Semester – Protein and Enzyme Practice questions      answers
               

 

PART 1.

 

1.   A 0.1 ml aliquot (i.e., a small fraction) of a 25.0 ml homogenate (total volume) of a chicken’s whole liver  (weighing 10.0 gm) is shown to have an Absorbance of 0.50 units.  Using the BSA (bovine serum albumin) Standard Protein curve below, how much protein is present in the total homogenate?

 

 

 

 

 

2.   If that same 0.1 ml aliquot of liver homogenate above converts 48 µmoles of glucose (substrate) to glucose-6-P (product) in 8 minutes then the total number of units of enzyme activity present in the chicken’s liver is?

 

 

3.   The specific activity of the enzyme (in question #2) from these chicken livers which converts glucose to glucose-6-P is?

 


 

 

 

PART 2.

 

4.  Succinate dehydrogenase is an enzyme that oxidizes succinate to fumerate. Malonic acid is
     a competitive inhibitor of the enzyme SDH. A mitochondrial sample of SDH gave the
     following data.


       By careful examination of the data determine the probable the Km for SDH is?

 

rate of NATIVE reaction                   concentration of succinate      

  2.0   units                             1.0  mmoles of starch

  7.5   units                            4.0  mmoles of starch

13.0   units                            9.1  mmoles of starch

15.05 units                          15.2   mmoles of starch

14.95 units                           25.1  mmoles of starch

15.0   units                           40.2  mmoles of starch

 

5.  From the data on SDH above (question 4), what would be the Vmax at high substrate
     concentrations for this enzyme in the presence of the competitive inhibitor malonic acid?
 

 

 

The next 4 questions relate to the figure to the below.  You have isolated two kinase enzymes (A and B) and are studying them. Enzyme A converts glucose + ATP <---> glucose-6P & enzyme B converts glucose + ATP <---> glucose-1-P.  You first measure the rate of reaction glucose + ATP <---> glucose-6P in the presence of a constant amount of enzyme A and varying amounts of substrate glucose.  You then measure the rate of the reaction glucose + ATP <---> glucose-1-P in the presence of a constant amount of enzyme B and varying amounts of glucose.  The number of enzyme A and B molecules per ml are exactly the same in the two Experiments.  You plot the results (see figure to the below). 

 

 

 

1.   The Vmax for enzyme B is closest to? 

 

2.   The Km for enzyme A is closest to which of the following?        

 

3.   Which enzyme, A or B, has the greater affinity for its own substrate?

 

4.  If enzyme A were subjected to non-competitive inhibition by the drug insulineride, you would
     expect the Vmax to ?
     


 

 

PART 3.

 

In the enzyme purification table below fill-in the question marks (???) and determine the amount of purification of our enzyme at the steps indicated.

 

 

Purification table for "cellbiolase" a new enzyme
 

STEP

Fraction Volume
(ml)

Total
 Protein

(mg)

Activity
(units)

Specific Activity
(units/mg protein)

1.  Homogenate
      supernatant

1,00

1,000

10,000

???

2.  Precipitate
 
from supernatant is
 dissolved in buffer

200

100

???

50

3. Ion-exchange chromatography

40

400

80,000

???

What level of increase is the purification from step 2 to 3?

4. Gel filtration

20

100

60,000

600

5. Affinity    chromatography

6

3

75,000

???*

 

What level of increase is the purification from step 1 to 5?