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Asparaginase

This document describes a spectrophotometric method for determining the activity of asparaginase (EC 3.5.1.1) by measuring the amount of ammonia released from asparagine. The method involves incubating asparaginase with asparagine, then adding trichloroacetic acid to stop the reaction and ammonia color reagent to detect ammonia. By comparing to a standard curve of known ammonia concentrations, the amount of ammonia released is used to calculate the activity of asparaginase in units per milliliter.

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67 views

Asparaginase

This document describes a spectrophotometric method for determining the activity of asparaginase (EC 3.5.1.1) by measuring the amount of ammonia released from asparagine. The method involves incubating asparaginase with asparagine, then adding trichloroacetic acid to stop the reaction and ammonia color reagent to detect ammonia. By comparing to a standard curve of known ammonia concentrations, the amount of ammonia released is used to calculate the activity of asparaginase in units per milliliter.

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Enzymatic Assay of ASPARAGINASE (EC 3.5.1.1) PRINCIPLE: L-Asparagine + H2O Asparaginase> L-Aspartate + NH3 CONDITIONS: T = 37C, pH = 8.

6, A436nm, Light path = 1 cm METHOD: Spectrophotometric Stop Rate Determination REAGENTS: A. 50 mM Tris Buffer, pH 8.6 at 37C (Prepare 100 ml in deionized water using Trizma Base, Prod. No. T-1503. Adjust to pH 8.6 at 37C with 1 M HCl.) 189 mM L-Asparagine Solution (Prepare 10 ml in deionized water using L-Asparagine, Anhydrous, Prod. No. A-0884.) 6 mM Ammonium Sulfate Standard Solution ((NH4)2SO4 Std) (Prepare 100 ml deionized water using Ammonium Sulfate, Grade I, Prod. No. A-5132.) 1.5 M Trichloroacetic Acid (TCA) (Prepare 10 ml in deionized water using Trichloroacetic Acid, 6.1 N Solution, Stock No. 490-10.) Ammonia Color Reagent (Use Nessler's Reagent, Aldrich Stock No. 34,518-8.) Asparaginase Enzyme Solution (Immediately before use, prepare a solution containing 2.0 - 4.0 units/ml of Asparaginase in cold deionized water.)

B.

C.

D.

E.

F.

SSASPA03 Revised:

Page 1 of 4
09/12/97

Enzymatic Assa y of ASPARAGINASE (EC 3.5.1.1) PROCEDURES: Step 1: Pipette (in milliliters) the following reagents into suitable tubes: Test Reagent A (Buffer) Reagent B (L-Asparagine Soln) Reagent C ((NH4)2SO4 Std) Deionized Water 1.00 0.10 ---0.90 Blank 1.00 0.10 ---0.90 Std 1 1.00 ---0.25 0.85 Std 2 1.00 ---0.50 0.60 1.00 ---1.00 0.10 Std Std 3 1.00 ------1.10 Blank

Equilibrate to 37C. Then add: Reagent F (Enzyme Solution)

0.10

----

----

----

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Immediately mix by inversion and incubate at 37C for 30 minutes. Then add: Reagent D (TCA) Reagent F (Enzyme Solution) 0.10 ---0.10 0.10 0.10 ---0.10 ---0.10 ---0.10 ----

Mix by inversion. Centrifuge for 2 minutes to clarify. Step 2: Pipette (in milliliters) the following reagents into suitable containers: Deionized Water Supernatant (from Step 1) Reagent E (Ammonia Color Reagent) 4.30 0.20 0.50 4.30 0.20 0.50 4.30 0.20 0.50 4.30 0.20 0.50 4.30 0.20 0.50 4.30 0.20 0.50

Immediately mix by inversion and after 1 minute record the A436nm for Standards, Tests, and Blanks.

SSASPA03 Revised:

Page 2 of 4
09/12/97

Enzymatic Assay of ASPARAGINASE (EC 3.5.1.1) CALCULATIONS: Standard Curve: r A436nm Standard = A436nm Standard - A436nm Standard Blank Prepare a standard curve by plotting the rA 436nm of the Standard versus Ammonia (NH3) concentration. Note that 1 mole of Ammonium Sulfate corresponds to 2 moles of Ammonia, therefore a 6 mM Ammonium Sulfate standard is equivalent to a 12 mM ammonium standard. Sample Determination: rA436nm Test = A436nm Test - A436nm Test Blank Determine the moles of NH3 liberated using the standard curve. (mole of NH3 liberated)(2.20) Units/ml enzyme = (0.2)(30)(0.1) 2.20 = Volume of Step 1 0.2 = Volume of Step 1 used in Step 2 30 = Time of assay in minutes 0.1 = Volume (in milliliters) of enzyme used units/ml enzyme Units/mg solid = mg solid/ml enzyme units/ml enzyme Units/mg protein = mg protein/ml enzyme UNIT DEFINITION: One unit will liberate 1.0 mole of ammonia from L-asparagine per minute at pH 8.6 at 37C. FINAL ASSAY CONCENTRATION: In a 2.20 ml reaction mix, the final concentrations are, 23 mM Tris, 8.6 mM L-asparagine and 0.2 - 0.4 units of asparaginase.

SSASPA03 Revised:

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09/12/97

Enzymatic Assay of ASPARAGINASE (EC 3.5.1.1) REFERENCES: Shirfrin, S., Parrott, C.L. and Luborsky, S.W. (1974) Journal of Biological Chemistry 249, 1335-1340 NOTES: 1. 2. This assay is based on the cited reference. Where Sigma Product or Stock numbers are specified, equivalent reagents may be substituted.

This procedure is for informational purposes. For a current copy of Sigmas quality control procedure contact our Technical Service Department.

SSASPA03 Revised:

Page 4 of 4
09/12/97

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