DiaPharma Group, Inc.

For research use only.

S-2444 is a chromogenic substrate for urokinase. The substrate has been used for the determination of:

1. Urokinase in purified preparations
2. Urokinase inhibitors in plasma
3. Urokinase activity in urine

Reagent -- Composition, Packaging, Storage and Stability

Each vial contains chromogenic substrate S-2444 25 mg and mannitol 40 mg added as a bulking agent. Substance: Stable until expiry date if stored at 2-8°C. Avoid exposure to light. The substance is hygroscopic and should be stored dry. Solution: 2 mmol/L in H2O is stable for at least 6 months at 2-8°C.

Chemistry

Chemical name: L-Pyroglutamyl-glycyl-L-argininep-Nitroaniline hydrochloride.

S-2444 structure

Formula:

Mol. wt.: 498.9

e316 nm: 1.27 · 104 mol-1 · L · cm-1

Solubility: > 10 mmol/L in H2O

Suitable stock solution: 2-3 mmol/L in H2O.

Principle

The method for the determination of activity is based on the difference in absorbance (optical density) between the pNA formed and the original substrate. The rate of pNA formation, i.e. the increase in absorbance per second at 405 nm, is proportional to the enzymatic activity and is conveniently determined with a photometer.

Kinetic data

Urokinase: Ploug U Km=9 · 10-5 mol/L, V=3.1 · 10-10 mol/min

CTA U Km=6 · 10-5 mol/L, V=1.3 · 10-10 mol/min

Determined at 37°C in 2.5 mL of 0.05 mol/L Tris buffer pH 8.8, I 0.05.

Standardization

An activity of DA/min= 0.05 (37°C) is obtained by using 0.3 mmol/L substrate and:

1. 25 Ploug U/mL of urokinase ( Leo, Copenhagen, Denmark)
2. 40 CTA U/mL of urokinase ( Abbott, Chicago, USA)