DiaPharma Group, Inc.

For research use only.

S-2238 is a chromogenic substrate for thrombin. The substrate has been used for the determination of:

1. Prothrombin in plasma
2. Antithrombin in plasma
3. Platelet factor 3 in plasma
4. Heparin in plasma

Reagent -- Composition, Packaging, Storage and Stability

Each vial contains chromogenic substrate S-2238 25 mg and mannitol 120 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: 1 mmol/L in H2O is stable for more than 6 months at 2-8°C.

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

Chemistry

Chemical name: H-D-Phenylalanyl-L-pipecolyl-Larginine-p-nitroaniline dihydrochloride.

S-2238 structure

Formula: H-D-Phe-Pip-Arg-pNA·2 HCl

Mol. wt.: 625.6

e316 nm: 1.27 . 104 mol-1 . L . cm-1

Solubility: > 10 mmol/L in H2O

Suitable stock solution: 1-2 mmol/L in H2O.

Principle

	Enzyme
H-D-Phe-Pip-Arg-pNA		H-D-Phe-Pip-Arg-OH+pNA

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.

S-2238 molecule

Model of S-2238 (green) fitted into the active site of thrombin

Kinetic Data

Human thrombin: Km = 0.7 ·10-5 mol/L

V = 1.7 · 10-7 mol/min · NIH-U

Bovine thrombin: Km = 0.9 ·10-5 mol/L

V = 2.2 · 10-7 mol/min · NIH-U

Both determined at 37°C in 2.5 mL 0.05 mol/L Tris buffer pH 8.3, I 0.15.

Standardization

An activity of DA/min = 0.05 (37°C) is obtained by using 0.1 mmol/L substrate and:

1. 0.03 NIH-U/mL of bovine thrombin (Roche or Parke-Davis)
2. 0.04 U/mL of human thrombin (MRC standard thrombin) Aprotinin (Trasylol®) may be added to the buffer in a concentration of 75 KIU/L in order to inhibit other activities than that of thrombin.

Note: For thrombin standardization against the MRCstandard the natural substrate fibrinogen, is recommended as the primary substrate. The clotting and amidolytic activities of degraded thrombins do not always develop in parallel.