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HomeEnzyme Activity AssaysEnzymatic Assay of Fumarase

Enzymatic Assay of Fumarase

1. Objective

To standardize a procedure for the enzymatic assay of Fumarase.

2. Scope

The scope of this procedure is for all products that have a specification for fumarase activity.

3. Definitions

3.1 Purified Water = water from a deionizing system, resistivity > or = 18MΩ•cm @ 25 ºC

3.2 Unit Definition - One unit will convert 1.0 μmol of L-malate to fumarate per minute at pH 7.6 at 25 ºC.

4. Discussion

L-Malate Fumarase > Fumarate + H2O

5. Responsibilities

Analytical services laboratory personnel should follow this procedure as written.

6. Safety

Refer to Safety Data Sheets (SDS) for hazards and appropriate handling precautions.

7. Procedure

7.1 CONDITIONS:
T = 25 °C, pH = 7.6, A240nm, Light path = 1 cm

7.2 METHOD:
Continuous Spectrophotometric Rate Determination

7.3 REAGENTS:

7.3.1 100mM Potassium Phosphate Buffer, pH 7.6 at 25 ºC (Buffer)
Prepare a 14 mg/mL solution in purified water using Potassium Phosphate, such as Product No. P5379. Adjust to pH 7.6 at 25 ºC with 1 N KOH.

7.3.2 50mM L-Malic Acid (Malate)
Prepare a 7 mg/mL solution in Reagent 7.3.1 (Buffer) using L-Malic Acid, such as Product No. M1000. Adjust to pH 7.6 at 25 ºC with KOH.

7.3.3 0.1% Bovine Serum Albumin (BSA)
Prepare a 1 mg/mL solution in purified water using Bovine Serum Albumin, such as Product No. A6003.

7.3.4 Fumarase Enzyme Solution (Enzyme)
Immediately before use, prepare a solution containing 1 unit/mL of fumarase in cold Reagent 7.3.3 (BSA). The enzyme solution MUST BE CLEAR AND COLORLESS before addition to the reaction mixture. Any cloudiness indicates the enzyme is not completely solubilized.

7.4 TEST METHOD

7.4.1 Pipette (in milliliters) the following reagents into suitable quartz cuvettes:

7.4.2 Mix by inversion and equilibrate to 25 ºC. Monitor the A240nm until constant, using a suitably thermostatted spectrophotometer. Then add:

Reagent 7.3.4 (Enzyme)

-
-
-
-

0.10

0.07

0.05

7.4.3 Immediately mix by inversion and record the increase in A240nm for approximately 10 minutes. Obtain the ΔA240nm/minute using the maximum linear rate.

7.5 CALCULATIONS

7.5.1

Units/ml =

(ΔA240nm/minute test - ΔA240nm/minute blank)(TV)(df)


(2.44)(SV)

where:
df = Dilution Factor
TV = Volume (in milliliters) of assay, differs for Test 1,2,3
2.44 = millimolar extinction coefficient of Fumarate at 240 nm
SV = Volume (in milliliters) of enzyme used, differs for Test 1,2,3

Units/mg protein =

Units/mL enzyme


mg protein/mL enzyme

7.6 FINAL ASSAY CONCENTRATION :
In a 3 mL reaction mix, the final concentrations are 96.7 mM potassium phosphate, 48.3 mM L-malic acid and 0.1 unit fumarase.

8. References

8.1 Bergmeyer, H.U., Gawehn, K., and Grassi, M. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H.U., ed) Volume I, 452, Academic Press, Inc., New York, NY.

Materials
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