Enzymatic Asparaginase Activity Assay

This assay protocol is suitable for the colorimetric/fluorometric detection of Asparaginase activity in bacteria, cell and tissue culture supernatants, plant samples, biological samples, and food samples using the Asparaginase Activity Assay Kit (MAK007). Asparaginase activity is determined by a coupled enzyme assay, which results in a colorimetric (570 nm)/fluorometric (λ_ex = 535/λ_em = 587 nm) product, proportional to the aspartate generated. One unit of Asparaginase is defined as the amount of enzyme that catalyzes the formation of 1.0 μmole of aspartate per minute at 25 °C.

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Reagents

Asparaginase Assay Buffer 25 mL
Product No. MAK007A

Fluorescent Peroxidase Substrate, in DMSO 0.2 mL
Product No. MAK007B

Substrate Mix 1 vl
Product No. MAK007C

Aspartate Enzyme Mix 1 vl
Product No. MAK007D

Conversion Mix 1 vl
Product No. MAK007E

Asparaginase Assay Positive Control 1 vl
Product No. MAK007F

Aspartate Standard, 100 mM 0.1 mL
Product No. MAK007G

Reagents and Equipment Required but Not Provided

96 well flat-bottom plate – It is recommended to use black plates with clear bottoms (Product No. M5686 or equivalent) for fluorescence assays and clear plates (Product No. M4436 or equivalent) for colorimetric assays.

Fluorescence or spectrophotometric multiwell plate reader

Preparation Instructions

Briefly centrifuge vials before opening. Use ultrapure water for the preparation of reagents. To maintain reagent integrity, avoid repeated freeze/thaw cycles.

Asparaginase Assay Buffer – Allow buffer to come to room temperature before use.

Fluorescent Peroxidase Substrate – Allow reagent to come to room temperature before use. Mix well by pipetting, then aliquot and store, protected from light and moisture, at –20 °C. Upon thawing, the Fluorescent Peroxidase Substrate is ready-to-use in the colorimetric assay.

For the fluorescence assay, dilute an aliquot of the Fluorescent Peroxidase Substrate 5 to 10-fold with Asparaginase Assay Buffer, just prior to use. This will reduce the background of the fluorescence assay.

Substrate Mix – Reconstitute in 0.5 mL of water. Mix well by pipetting, then aliquot and store at –20 °C. Use within two months of reconstitution.

Aspartate Enzyme Mix and Conversion Mix – Reconstitute each in 220 μL of Asparaginase Assay Buffer. Mix well by pipetting, then aliquot and store at –20 °C. Use within two months of reconstitution.

Asparaginase Assay Positive Control – Reconstitute in 100 μL of Asparaginase Assay Buffer. Mix well by pipetting, then aliquot and store at –20 °C. Use within two months of reconstitution.

Storage/Stability

The kit is shipped on wet ice. Storage at –20 °C, protected from light, is recommended.

Procedure<

All samples and standards should be run in duplicate.

Aspartate Standards for Colorimetric Detection
Dilute 10 μL of the 100 mM (100 nmole/μL) Aspartate Standard Solution with 990 μL of Asparaginase Assay Buffer to prepare a 1 mM (1 nmole/μL) standard solution. Add 0, 2, 4, 6, 8, and 10 μL of the 1 mM Aspartate standard solution into a 96 well plate, generating 0 (blank), 2, 4, 6, 8, and 10 nmole/well standards. Add Asparaginase Assay Buffer to each well to bring the volume to 50 μL.

Aspartate Standards for Fluorometric Detection
Dilute 10 μL of the 100 mM (100 nmole/μL) Aspartate Standard Solution with 990 μL of Asparaginase Assay Buffer to prepare a 1 mM (1 nmole/μL) standard solution. Dilute 10 μL of the 1 mM standard solution with 90 μL of Asparaginase Assay Buffer to generate a 0.1 mM (0.1 nmole/μL) standard solution. Add 0, 2, 4, 6, 8, and 10 μL of the 0.1 mM Asparate standard solution into a 96 well plate, generating 0 (blank), 0.2, 0.4, 0.6, 0.8, and 1.0 nmole/well standards. Add Asparaginase Assay Buffer to each well to bring the volume to 50 μL.

Sample Preparation
Both the colorimetric and fluorometric assays require 50 μL of sample for each reaction (well).

Tissue or cells should be rapidly homogenized with 4 volumes of Asparaginase Assay Buffer. Centrifuge at 15,000 x g for 10 minutes to remove insoluble materials. Bring samples to a final volume of 50 μL with Asparaginase Assay Buffer.

Serum samples can be directly added to wells. Add 1–50 μL samples into wells of a 96 well plate. Bring samples to final volume of 50 μL with Asparaginase Assay Buffer.

For unknown samples, it is suggested to test several sample volumes to make sure the readings are within the standard curve range.

For the positive control (optional), add 5 μL of the Asparaginase Assay Positive Control to wells. Adjust well volume to 50 μL with water.

Aspartate, oxaloacetate, and pyruvate in the samples will generate a background signal. To remove the effect of background, a sample blank may be set up by omitting the Aspartate Enzyme Mix. The blank readings can then be subtracted from the sample readings.

Assay Reaction
1. Set up the Reaction Mixes according to the scheme in Table 1. Prepare enough Reaction Mixes for the number of samples, positive controls, and standards to be performed.

2. Add 50 μL of the appropriate Reaction Mix to each well. Mix well using a horizontal shaker or by pipetting.

3. After 2–3 minutes, take the initial measurement (T_initial). For colorimetric assays, measure the absorbance at 570 nm (A₅₇₀)_initial. For fluorometric assays, measure fluorescence intensity (FLU_initial, λ_ex = 535/λ_em = 587 nm).

4. Incubate the plate at 25 °C taking measurements every 5 minutes. Protect the plate from light during the incubation.

5. Continue taking measurements until the value of the most active sample is greater than the value of the highest standard. At this time the most active sample is near or exceeds the end of the linear range of the standard curve.

6. The final measurement for calculating the enzyme activity would be the penultimate reading or the value before the most active sample is near or exceeds the end of the linear range of the standard curve. The time of the penultimate reading is T_final.

7. Calculate the change in measurement from T_initial to T_final.

>ΔA₅₇₀ = (A₅₇₀)_final – (A₅₇₀)_initial

or

ΔFLU = (FLU_final) – (FLU_initial)

Note: It is essential the initial and final measurements fall within the linear range of the reaction.

Results

Calculations
Correct for the background by subtracting the value obtained for the 0 (blank) standard from all readings. Plot the aspartate standard curve.

Note: A new standard curve must be set up each time the assay is run.

Compare the Δmeasurement value (ΔA₅₇₀ or ΔFLU) of each sample to the standard curve to determine the amount of aspartate generated (B) between T_initial and T_final.

The Asparaginase activity of a sample may be determined by the following equation:

Asparaginase Activity = (B x Sample Dilution Factor)/[(T_final – T_initial) x V]

B = Amount (nmole) of aspartate generated between T_initial and T_final
T_initial = Time of first reading in minutes.
T_final = Time of second reading in minutes.
V = sample volume (mL) added to well.

Asparaginase activity is reported as nmole/min/mL = milliunit/mL, where one unit of Asparaginase is defined as the amount of enzyme that catalyzes the formation of 1.0 mmole of aspartate per minute at 25 °C.