Enzymatic Assay of Elastase (EC 3.4.21.36)

Description

This procedure may be used for Elastase products using SucAla3-pNA as the substrate.

The continuous spectrophotometric rate determination (A410, Light path = 1 cm) is based on the following reaction:

where:
SucAla3-pNA = N-Succinyl-Ala-Ala-Ala-p-nitroanilide
SucAla3 = N-Succinyl-Ala-Ala-Ala
p
NA = p-Nitroanilide

Unit Definition – One unit of Elastase will hydrolyze 1.0 µmole of N-succinyl-L-Ala-Ala-Ala-p-nitroanilide per minute at pH 8.0 at 25 °C.

Precautions

Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Reagents and Equipment Required

Trizma® base (Catalog No. T1503)
N-Succinyl-Ala-Ala-Ala-p-nitroanilide (Catalog No. S4760)

Preparation Instructions

Use ultrapure water (≥18 MΩxcm resistivity at 25 °C) for the preparation of reagents.

Buffer (100 mM Tris HCl, pH 8.0 at 25 °C) – Prepare a 12.1 mg/mL solution of Trizma base (Catalog No. T1503) in ultrapure water. Adjust the pH to 8.0 at 25 °C with 1 M HCl.

Substrate Solution (4.4 mM SucAla3-pNA Solution) – Prepare 2 mg/mL solution of N-Succinyl-Ala-Ala-Ala-p-nitroanilide (Catalog No. S4760) in Buffer.

Enzyme Solution (Elastase) – Immediately before use, prepare a solution containing 0.2–0.5 unit/ml of Elastase in cold (2–8 °C) buffer.

Procedure

In a 3.00 ml reaction mix, the final concentrations are 96.7 mM Trizma, 0.29 mM N-Succinyl-Ala-Ala-Ala-p-nitroanilide, and 0.02–0.05 unit of Elastase.

1.     Pipette the following reagents into suitable cuvettes:

 

Reagent Test (ml) Blank (ml)
Buffer 2.70 2.80
Substrate Solution 0.20 0.20


2.     Mix by inversion and equilibrate to 25 °C. Then add:

 

Reagent Test (ml) Blank (ml)
Enzyme Solution 0.10


3. Immediately mix by inversion and record the increase in A410 for ~5 minutes. Obtain the ΔA410/minute using the maximum linear rate for both the Test and Blank using a minimum of 4 data points over a one minute time interval.

Results

Calculations

1.

Units/ml enzyme = (ΔA410/min Test – ΔA410/min Blank) (3.00) (df)
(8.8) (0.10)


where:
3.00 = Total volume (ml) of assay
df = Dilution factor
8.8 = Millimolar extinction coefficient of p-Nitroaniline at 410 nm at pH 8.0
0.1 = Volume (ml) of Enzyme Solution used


2.

Units/mg solid = units/ml enzyme
mg solid/ml enzyme

Materials

     

References

  • Bieth, J. et al., Biochemical Medicine, 11, 350-357 (1974).

 

Trizma is a registered trademark of Sigma-Aldrich Co. LLC.

02/14-1

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