Determination of urokinase activity with Chromogenic S-2444
Measurement Principle
The urokinase activity is determined by its amidolytic effect on the chromogenic substrate pyro-Glu-Gly-Arg-pNA (chromogenic substrate S-2444). 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 DA/min (or absorbance) and the urokinase activity is linear in the range 5-40 Ploug or CTA units. The urokinase concentration should preferably be given in units of substrate hydrolyzing activity, but may be calculated by using standards prepared from a standard urokinase preparation. The amidolytic activity, however, does not necessarily parallel the fibrinolytic activity for different urokinases.
Urokinase
pGlu-Gly-Arg-pNA+H2O → pGlu-Gly-Arg-OH + pNA
Reagents
- Chromogenic S-2444, 25 mg Art. No. S820357
Reconstitute the chromogenic substrate S-2444 (MW: 498.9) with 16.7 ml of distilled water. - Urokinase standard
The urokinase standard is dissolved in or diluted with Solvent (Reagent 3) to a concentration of 400 units/ml (Ploug or CTA units). The dilution is stable for one day at 2-8°C. - Solvent
Distilled water containing 5 g/l of Carbowax 6000 (Union Carbide, NY, USA). - Tris Buffer, pH 8.8 (25°C)Tris 6.1 g (50 mmol/l)
NaCl 2.2 g (38 mmol/l)
Distilled water 800 mlAdjust the pH to 8.8 at 25°C by adding an appropriate amount (approx. 12 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 2-8°C. Note: Although the substrate is quite selective, there may be a risk for influence of other proteases if the preparation is heavily contaminated. The addition of Trasylol (aprotinin), 10 KIU/ml, to the buffer may in such cases be favorable. - Acetic acid 20%
Acetic acid is used in the acid-stopped method.
Sample
The urokinase is dissolved in or diluted with Solvent (Reagent 3) to a concentration of approximately 400 units/ml (Ploug or CTA units) By using commercially available urokinase (Leo or Abbott) it was found that the dilution was stable for at least one day when kept at 2-8°C. Note: if the urokinase preparation is contaminated with proteolytic enzymes, Trasylol (aprotinin) may be added to a concentration of 10 KIU/ml in order to increase the stability.
Standardization
40 units: Use the urokinase standard 400 units/ml (Reagent 2). 5 units: Use the urokinase standard 400 units/ml (Reagent 2) diluted 1:8 with buffer (Reagent 4).
Standard curve
The urokinase standard 400 units/ml (Reagent 2) is further diluted according to the table below:
| Plong or CTA Units | Urokinase standard (400 units/ml) µl | Solvent µl |
|---|---|---|
| 5 | 100 | 700 |
| 10 | 100 | 300 |
| 20 | 200 | 200 |
| 30 | 300 | 100 |
| 40 | 400 | – |
Method
Initial rate method
| Buffer | 800 µl |
| Incubate at 37°C | 5-10 min |
| Urokinase samples/standards | 100 µl |
| Incubate at 37°C | 1-2 min |
| Substrate (37°C) | 100 µl |
| Mix |
Transfer 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 | Sample blank |
|---|---|---|
| Buffer | 800 µl | 800 µl |
| Incubate at 37°C | 5-10 min | – |
| Urokinase samples/standards | 100 µl | 100 µl |
| Incubate at 37°C | 1-2 min | – |
| Substrate (37°C) | 100 µl | – |
| Incubate at 37°C | 5 min | – |
| Acetic acid | 100 µl | 100 µl |
| Mix | yes | yes |
| Incubate at 37°C | 5 min | – |
| Substrate (37°C) | – | 100 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
Plot ΔA/min or A for the standards against their known urokinase activity. Calculate the urokinase activity of the sample in Ploug or CTA units. By multiplying the results with 10 the concentration in units/ml is obtained. The urokinase activity can also be calculated from the following formulas:
Initial rate method:
µkat/l = ΔA/min x 17.4
U/l = ΔA/min x 1042
Acid stopped method:
µkat/l = A x 3.8
U/l = A x 229
Bibliography
- Claeson G et al. Methods for determination of prekallikrein in plasma, glandular kallikrein and urokinase. Haemostasis 7, 76-78 (1978).
- Paar D and Marhuln D. Spectrophotometric determination of urokinase in urine after gel filtration, using the chromogenic substrate S-2444. J Clin Chem Clin Biochem 18, 557-562 (1980).
- Friberger P: Chromogenic Peptide Substrates. Scand J Clin Lab Invest 42, suppl. 162, 55 (1982).
- Philo R D and Gaffney P J: Relative potencies of different molecular weight forms of urokinase. In: Progress in Chemical Fibrinolysis and Thrombolysis. Vol. V Ed Davidson J F, Nilsson I M and Åstedt B, 220-222 (1980).
- Millar WT and Smith JF. Comparative study of different urokinase preparations. Thromb Res 30, 425-429 (1983). Higazi AA and Mayer M. In vitro inhibition of urokinase by penicillins. Thromb Haemost 60, 305-307 (1988).
- Kebabian PR and Henkin J. A chromogenic enzymatic assay capable of detecting prourokinase-like activity material in plasma. Thromb Res 65, 401-407 (1992).
- Bhargava K and Ando HY. Immobilization of active urokinase on albumin microspheres: use of a chemical dehydrant and process monitoring. Pharm Res 9, 776-781 (1992).