Determination of granulocyte elastase activity with Chromogenic Substrate S-2484
Measurement Principle
The elastase activity is determined by its amidolytic effect on the substrate pyro-Glu-Pro-Val-pNA (S-2484). The rate at which p-nitroaniline (pNA) is released is measured photometrically at 405 nm. This can be followed on a recorder (initial rate method) or read after stopping the reaction with acetic acid (acid stopped method). The correlation between the change in absorbance per minute (ΔA/min) or absorbance (A) and the granulocyte elastase activity is linear in the 0.1-1.5 µkat/l or 6-90 U/l range. The amidolytic activity does not necessarily parallel the elastolytic activity for different elastase preparations.
Granulocyte elastase
pGlu-Pro-Val-pNA + H2O → pGlu-Pro-Val-OH + pNA
Reagents
- Chromogenic Substrate S-2484, 25 mg Art. No. 82 08 86
Reconstitute the substrate S-2484 (MW: 445.5) with 7 ml of DMSO. One volume of this stock solution is diluted with 3 volumes of distilled water. - Tris Buffer, pH 8.3 (25°C)
Tris 12.1 g (100 mmol/l)
NaCl 52.6 g (960 mmol/l)
Distilled water 800 ml
Adjust the pH to 8.3 at 25°C by adding about 50 ml of 1 mol/l HCl. Fill up to 1000 ml with distilled water. The buffer, if not contaminated, will remain stable for two months at 2 to 8°C - Acetic acid 20%
Acetic acid is used in the acid-stopped method.
Sample
The sample containing granulocyte elastase is dissolved in or diluted with distilled water, saline or buffer to an activity of 0.1-1.5 µkat/l which approximately corresponds to a concentration of 0.5-7.5 mg/l of a rather pure enzyme. If the sample is a pure protein, it is advisable to use 0.1% Carbowax 6000 (Union Carbide, NY) or 1% albumin (previously checked for amidolytic activity) to avoid adsorption to surfaces.
Method
Initial rate method
| Buffer | 200 µl |
| Incubate at 37°C | 3-4 min |
| Elastase sample | 200 µl |
| Mix and incubate at 37°C | 2-3 min |
| Substrate | 200 µl |
Transfer the sample immediately to a 1 cm semi-microcuvette (preheated to 37°C) for measurement of the absorbance change in a photometer at 405 nm and at 37°C. Calculate ΔA/min.
| Acid stopped method | Sample | Blank |
|---|---|---|
| Buffer | 200 µl | 200 µl |
| Incubate at 37°C | 3-4 min | – |
| Elastase sample | 200 µl | µl |
| Mix and incubate at 37°C | 2-3 min | – |
| Substrate (37°C) | 200 µl | – |
| Mix and incubate at 37°C | 3 min | – |
| Acetic acid 20% | 200 µl | 200 µl |
| Mix | yes | – |
| Substrate | – | 200 µl |
| Mix | – | yes |
Read the absorbance (A) of the sample against a water or sample blank in a photometer at 405 nm. The color is stable for at least 4 hours.
Calculation
Calculate the elastase activity of the sample from the formulas:
Initial rate method:
µkat/l = 5.19 x ΔA/min
U/l = 311 x ΔA/min
Acid stopped method:
µkat/l = 2.31 x A
U/l = 138 x A
Bibliography
- Kramps JA et al. L-Pyroglutamyl-L-prolyl-L-Valine-p-nitroanilide, a highly specific substrate for granulocyte elastase. Scand J Clin Lab Invest 43, 427-432, (1983).
- Tanaka H et al. A sensitive and specific assay for granulocyte elastase in inflammatory tissue fluid using L-pyroglutamyl-L-prolyl-L- valine-p-nitroanilide. Clin Chim Acta 187, 173-180 (1990).
- Liu R et al. Leukocyte functions in 2 cases of Papillon-Lefevre syndrome. J Clin Periodontol 27, 69-73 (2000).