Assignment 3

Question 1. Fill-in-the-blank (1 point—0.5 points/blank)

In the biuret assay, the copper ion of the reagent reacts with ____ bonds of protein to give a violet color. The tube with a known amount of protein contains the substance ______.

Question 2. Fill-in-the-blank (1 point—0.5 points/blank)

Based on the results of the biuret assay:

The unknown substance that has the least amount of protein is in tube _____.

The unknown substance in tube 4 contains approximately ____ mg of protein. (Give your best estimate).

Question 3. File upload (2 points)
Record the data from the glucose assay below (use this spectrophotometer to measure absorption). Be sure to set the spectrophotometer to zero when recording absorption from the 0 tube. Then type the number of each test tube (1 through 7) in the white box and click "measure" to get the absorption of the solution in each tube.

Then graph the standard curve with the on-line plotter, using a linear regression. Be sure to label the x and y axes on this and all future graphs. NOTE: tubes 6 and 7 are not part of the standard curve—they are your solutions that contain unknown quantities of glucose.

Capture an image of the graph and table beneath it, and upload it to WebAssign. This graph is your standard curve: you will use it to calculate glucose amounts in your unknowns for questions 4 and 6.

Tube
0 / Tube 1 / Tube 2 / Tube 3 / Tube 4 / Tube 5 / Tube 6 / Tube 7
Glucose amount / 0 / 5 ug / 10 ug / 20 ug / 30 ug / 40 ug / ? / ?
Absorption
At 420 nm / 0

Question 4. Fill-in-the-blank (3 points).
Record the absorption of the unknown solutions (tubes 6 and 7). Using the regression line of your standard curve, calculate the amount of glucose in the unknown solutions.

The amount of glucose in tube 6 is ____ ug and the amount in tube 7 is ____ ug.

Show your work for calculation of these values. (Your calculations must be shown to receive full credit for this question.)

Question 5. Multiple-choice (1 point)

The mouse intestine was flushed before homogenization in order to:

Remove the enzyme maltase from the intestine

Remove the substrate maltose from the intestine

Remove blood from the intestine

Remove feces from the intestine

None of the above

Question 6. File upload (2 points)
Use this spectrophotometer to measure and record the absorption of each solution. Be sure to set the spectrophotometer to zero when recording absorption from the 0 tube.

Then use your glucose standard curve to calculate amounts of glucose. Divide these values by incubation time to determine maltase activity. Complete the following table:

Tube
number / Incubation
Time (min) / Absorption / Glucose (ug) / Maltase activity
(ug glucose/min)
0 / 0
1* / 0
2 / 5
3 / 10
4 / 15
5 / 20
6 / 30

*This tube contains both homogenate and substrate, but was not incubated.
(TGO was added immediately after the substrate).

Make a line graph of glucose amount (ug) vs. incubation time using the on-line plotter. Capture an image of the plotter, including both graph and plotter table, and submit it to WebAssign.

Question 7. True/False (2 points—0.5 points/blank)
Refer to the data in the above table to answer the following questions

_____ Maltase activity measured in the time curve assay was approximately 2.2 ug glucose/min.

_____ When glucose is plotted against incubation time, the ideal graph should be a straight line.

_____ When maltase activity (ug glucose/min) is plotted against time, the ideal graph should be a straight line.

_____ In a “perfect” assay, maltase activity (ug glucose/min) will be quite different at each incubation time.

Question 8. File upload (2 points)
Use this spectrophotometer to measure and record the absorption of each solution.

Be sure to set the spectrophotometer to zero when recording absorption from the 0 tube.

Tube
number / 0 / 1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9
Maltose
concentration / 0 / 1.0
mM / 2.5
mM / 5.0
mM / 10
mM / 25
mM / 50
mM / 75 mM / 100
mM / 150 mM
Absorption of solutions / 0

Now use the on-line plotter to graph absorption vs. substrate concentration. Make a scatter plot—if you want to add lines, make sure to connect the points (do not make a line regression).
Capture an image of the plotter, including both graph and plotter table, and submit it to WebAssign.

Questions 9 and 10 refer to the substrate concentration graph.

Question 9. Multiple-choice (1 point)

Which of the following best describes the effect of substrate concentration on maltase activity?

As substrate concentration increases, maltase activity increases in proportion.

As substrate concentration increases, maltase activity increases almost linearly at first, but levels off at higher concentrations.

As substrate concentration increases, maltase activity increases almost linearly at first, then decreases at higher concentrations.

As substrate concentration increases, maltase activity increases slowly and first, then more rapidly at higher concentrations

Substrate concentration has no effect on maltase activity.

Question 10. Multiple-choice (1 point)

Which of the following changes in the maltase assay would be most likely to change the graph in question 8 into a straight line?

Increasing all amounts of substrate

Using a shorter incubation time

Using a larger amount of intestinal homogenate

Using a smaller amount of intestinal homogenate

Graphing ug glucose (instead of absorption) on the Y axis

Question 11. File upload (2 points)

Use the on-line plotter to construct a graph that displays the effect of pH on maltase activity. Capture an image of the plotter, including both graph and plotter table, and submit it to WebAssign.

Question 12. File upload (2 points)
Use the on-line plotter to construct a graph that displays the effect of temperature on maltase activity. Capture an image of the plotter, including both graph and plotter table, and submit it to WebAssign.

Question 13. Multiple-choice (1 point)

Based on the data in your graph, the pH optimum for maltase is:

A neutral pH

A slightly acidic pH

A slightly basic pH

A pH of 10

Maltase activity is the same at all pH values

Question 14. Multiple-choice (1 point)

Based on the temperature data in your graph, which of the following is a true statement?

Maltase has higher activity at 10°C than at 20°C.

Maltase has no activity at 4°C.

Maltase has no activity when the enzyme is boiled before assay.

The optimal temperature for maltase activity is 20°C.

All of these statements are true.

Question 15. Multiple-choice (1 point)

Study your graph of copper concentration vs. absorption. The concentration of copper ion that produces a 50% inhibition of maltase activity is approximately:

0.1%

0.4%

2.0%

4.0%

10.0%

Question 16. Matching (5 points)
Complete the table, then answer the matching question in WebAssign.

Tube number / Correct label for tubes (A-E)
1
2
3
4
5