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:
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.
Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.
Trizma® base (Catalog No. T1503)
N-Succinyl-Ala-Ala-Ala-p-nitroanilide (Catalog No. S4760)
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.
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:
2. Mix by inversion and equilibrate to 25 °C. Then add:
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.
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