Determination of urokinase activity with Chromogenic S-2444
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.
pGlu-Gly-Arg-pNA+H2O → pGlu-Gly-Arg-OH + pNA
- 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.
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.
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.
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).
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|
Initial rate method
|Incubate at 37°C||5-10 min|
|Urokinase samples/standards||100 µl|
|Incubate at 37°C||1-2 min|
|Substrate (37°C)||100 µl|
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|
|Incubate at 37°C||5 min||–|
|Substrate (37°C)||–||100 µl|
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.
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
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