Chromogenic Substrates

 

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CHROMOGENIC SUBSTRATES UNIVERSITY | METHODS | UROKINASE METHOD


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 hydrolysing 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

Urokinase
pGlu-Gly-Arg-OH + pNA

Reagents

  1. Chromogenic S-2444, 25 mg    Art. No. S820357
    Reconstitute the chromogenic substrate S-2444 (MW: 498.9) with 16.7 ml of distilled water.

  2. 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 2-8C.

  3. Solvent
    Distilled water containing 5 g/l of Carbowax 6000 (Union Carbide, NY, USA).

  4. Tris Buffer, pH 8.8 (25C)

    Tris 6.1 g (50 mmol/l)
    NaCl 2.2 g (38 mmol/l)
    Distilled water 800 ml

Adjust the pH to 8.8 at 25C 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 2-8C. 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 favourable.

  1. 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 2-8C. 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.

Standardisation
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)

Solvent
ml ml

5

100 700
10 100 300
20 200 200
30 300 100
40 400 -

Method

Initial rate method

Buffer

800 l

Incubate at 37C

5-10 min

Urokinase samples/standards

100 l

Incubate at 37C

1-2 min

Substrate (37C)

100 l

Mix

Transfer sample immediately to a 1 cm semi-microcuvette (preheated to 37C) for measurement of the absorbance change in a photometer at 405 nm and at 37C, calculate DA/min.

Acid stopped method

Sample Sample blank

Buffer

800 l 800 l

Incubate at 37C

5-10 min -

Urokinase samples/standards

100 l 100 l

Incubate at 37C

1-2 min -

Substrate (37C)

100 l -

Incubate at 37C

5 min -

Acetic acid

100 l 100 l

Mix

yes yes

Incubate at 37C

5 min -

Substrate (37C)

- 100 l

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
DA/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:
kat/l =
DA/min x 17.4
U/l =
DA/min x 1042 

Acid stopped method:
kat/l = A x 3.8
U/l = A x 229

Bibliography

  1. Claeson G et al. Methods for determination of prekallikrein in plasma, glandular kallikrein and urokinase. Haemostasis 7, 76-78 (1978).

  2. 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).

  3. Friberger P: Chromogenic Peptide Substrates. Scand J Clin Lab Invest 42, suppl. 162, 55 (1982).

  4. 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 stedt B, 220-222 (1980).

  5. 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).

  6. Kebabian PR and Henkin J. A chromogenic enzymatic assay capable of detecting prourokinase-like activity material in plasma. Thromb Res 65, 401-407 (1992).

  7. 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).

 

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