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
pNA = 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

References

1. Bieth J, Spiess B, Wermuth CG. 1974. The synthesis and analytical use of a highly sensitive and convenient substrate of elastase. Biochemical Medicine. 11(4):350-357. https://doi.org/10.1016/0006-2944(74)90134-3