Spectrophotometry / Enzyme Assays

BIOLOGY 371

INDEPENDENT RESEARCH

WEEK 1

Spectrophotometry / Enzyme Assays

Objectives:

1. Learn spectrophotometric assay of isocitrate dehydrogenase.

2. Determine the effects of varying concentrations of enzyme.

3. Determine the effects of varying concentrations of substrate.

4. Determine the effects of varying concentrations of NADP.

5. Learn how to "manage" data using a specific graphing program.

ASSAY SOLUTION

Isocitrate Dehydrogenase
[pH 8.5 / 0.2 M Tris / 0.1437 mM NADP / 1 mM MgCl₂]
Tris HCl 7.32 gm
Tris Base 18.6 gm
MgCl₂ 203 mg
Water To 1 litre
When used, add 11 mg of NADP / 100 mL

SUBSTRATE CONCENTRATION

Isocitrate
11.9 mg in 2 mL of water
Use 10 µl / assay for final concentration of 0.023 mM.

ASSIGNMENTS: 1. Evaluate the above molarities for correctness. 2. Write a two page essay on spectrophotometry and its application to enzymology.

ASSAYS FOR NADP-ENZYMES

1. Routine assays are conducted in 1 mL cuvettes, at 30° C, in standard assay solution.
2. Turn on water bath [30° C], prepare appropriate volumes of assay solution [usually 50 - 100 mL]. Place in water bath.
3. Obtain bucket of ice, pipets, tips, substrate, wash bottle with distilled water, parafilm, clean cuvettes.
4. Prepare enzyme source and store on ice.
5. Turn on the spectrophotometer and water heater.
6. Set spectrophotometer at 340 nm.
7. Running a blank assay:

Add 1 mL of assay buffer to each of two 1 mL cuvettes.
Set ABS to 0.000.
Observe for three minutes.
ABS should remain at or near 0.000.

8. Running an enzyme blank assay:

Add 10 m l of enzyme prep to sample cuvette from Step 7.
Set ABS at 0.000.
Observe for 3 minutes.
If ABS changes, reset to 0.000 and observe for 3 more minutes.

9. Running an assay:

Add 1 mL of assay buffer to a clean cuvette.
Add 10 m l of enzyme prep.
Mix contents by covering with a [1 inch] square of parafilm and
inverting the cuvette.
Incubate in 30° C water bath for two minutes.
Clean the cuvette and insert into the spectrophotometer.
Read for 1 - 2 minutes to insure that no reaction is taking place.
Add 10 m l of substrate, mix, clean cuvette and re-insert into the
spectrophotometer.
Record activity on the recorder.

10. Calculation of initial velocity:

Draw a straight line that best fits the recording.
Determine the slope of this line at 1 ABS unit [= OD / minute].
Multiply OD / minute by 0.1613 to determine the initial velocity
of the reaction [V₀].

11. Calculation of Specific Activity:

Sp Ac = V0 / mg of protein in the reaction solution.

Figure 1. The activity of a reaction varies directly with the amount of enzyme added to the reaction mixture.

EFFECTS OF ENZYME CONCENTRATION ON ACTIVITY

ul of Enzyme Slope Mean Slope Activity*

2.5 0.467

0.387 0.415 0.067

0.391

5.0 1.25

1.11 1.17 0.189

1.15

10.0 1.29

1.41 1.30 0.211

1.22

15.0 1.75

1.60 1.784 0.288

2.00

20.0 2.92

2.66 2.17 0.436

2.52

* m g of NADP reduced per minute

Figure 2. The relationship between substrate concentration and activity is not linear.

Figure 3. The relationship between the concentration of NADP+ and activity is not linear.

Return to Schedule

ul of Enzyme	Slope	Mean Slope	Activity*
2.5	0.467
	0.387	0.415	0.067
	0.391

5.0	1.25
	1.11	1.17	0.189
	1.15

10.0	1.29
	1.41	1.30	0.211
	1.22

15.0	1.75
	1.60	1.784	0.288
	2.00

20.0	2.92
	2.66	2.17	0.436
	2.52