DiaPharma Group, Inc.

For research use only

S-2222 is a chromogenic substrate for Factor Xa. It is also very sensitive to trypsin. The substrate has been used for the determination of:

1. Factor X in plasma
2. Factor Xa in plasma
3. Factor Xa inhibitor in plasma
4. Heparin in plasma
5. Factor VIII in plasma
6. Coagulating enzyme from horseshoe crab plasma
7. Trypsin in duodenal fluid

The substrate is also sensitive to subtilisin, acrosin and Factor XIIa but insensitive to most other enzymes tested, e.g. Factor IXa, kallikrein (glandular and plasma) and papain-like enzymes.

Molecular wt: 741.3

Formula: Bz-Ile-Glu(g-OR)-Gly-Arg-pNA·HCl
R=H (50%) and R=CH3 (50%)

S-2222 structure

Reagent -- Composition, Packaging, Storage and Stability

Each vial contains chromogenic substrate S-2222 25 mg and mannitol 120 mg as a bulking agent.

Stability: Substance: Stable at 25°C for more than 3 years. The substance is somewhat hygroscopic and should be stored dry.

Solution: 4 mmol/L in H2O is stable for at least 6 months at 2 to 8°C.

Suitable stock 1-4 mmol/L in H2O. Vigorous shaking or solution: an ultrasonic bath is recommended for dissolution, which is slow.

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

Chemistry

Chemical name: N-Benzoyl-L-isoleucyl-L-glutamyl-glycyl-Larginine-p-nitroaniline hydrochloride and its methyl ester

Solubility:
6 mmol/L in H2O
2 mmol/L in Tris buffer (pH 8.3, I 0.25)

Principle

	Enzyme
Bz-lle-Glu-Gly-Arg-pNA		Bz-lle-Glu-Gly-Arg-OH+pNA

The method for the determination of activity is based on the difference in (yellow) 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

Factor Xa (bovine): Km=3. 10-4 mol/L. kcat=100 sec-1 in 37°C. Tris buffer pH 8.3, I 0.25
Trypsin (porcine): Km=2. 10-5 mol/L, kcat=280 sec-1 in 37°C. Tris buffer pH 9.0, I 0.25

Standardization

An activity of DA/min=0.05 (37°C) is obtained by using a substrate concentration of 2. kmand:

1. 0.1 nkat/mL of Factor Xa (Chromogenix) at pH 8.
2. Normal plasma diluted 1:150 and activated with 6 mg RVV (Sigma) per mL of the dilution.

The same activity is obtained by using 5. 10-13 mol/L of porcine trypsin.