DiaPharma Group, Inc.

For research use only

S-2366 is a chromogenic substrate for factor XI and activated protein C.The substrate can be used for the determination of purified enzyme preparations as well as of Protein C and FXI in plasma.

Reagent -- Composition, Packaging, Storage and Stability

Each vial contains chromogenic substrate S-2366 25 mg and mannitol 40 mg as a bulking agent. Stability: 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 more than 6 months at 2 to 8°C. Suitable stock solution: 2-3 mmol/L in H2O.

** Also available in bulk sizes (500+ mg) as a special order.

Chemistry

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

Formula: < Glu-Pro-Arg-pNA·HCl

S-2366 structure

Mol. wt: 539.0

e316 nm: 1.27· 104 mol-1· L· cm-1

Solubility: > 10 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

Protein C:

Km=2· 10-4 mol/L and kcat=80 sec-1 (The enzyme is assumed to be pure. Mol. wt. 62 000) Determined with RVV activated bovine Protein C in 0.05 mol/L Tris, pH 8.0, I 0.25 (NaCl) and 4mmol/L CaCl2 at 37°C.

Km=8 · 10-4 mol/L and kcat=160 sec-1. Determined with thrombintrombomodulin complex activated human Protein C in 0.05 mol/L Tris, pH 8.0, I 0.13 (NaCl) and 10 mmol/L CaCl2 at 25°C.

FXIa:

Km=4 · 10-4 mol/L and kcat @ 1000 sec-1 in 0.1 mol/L Phosphate buffer, pH 7.6, I 0.15 mol/L (NaCl) at 37°C.

Km=5.6 · 10-4 mol/L and kcat=350 sec-1 in 0.09 mol/L Tris, pH 8.3, 0.09 mol/L NaCl, 1 mg/mL of bovine serum albumin at room temperature.

Selectivity

S-2366 is also readily split by trypsin, thrombin, plasmin and tissue plasminogen activator. It is split by FXIIa, plasma kallikrein and FXa as well.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.

Hirudin Research Method