DiaPharma Group, Inc.

For research use only.

S-2266 is a chromogenic substrate for glandular kallikrein. The substrate has been used for the determination of:

1. Kallikrein activity in purified preparations
2. Kallikrein in urine
3. Kallikrein in saliva

Reagent -- Composition, Packaging, Storage and Stability

Each vial contains chromogenic substrate S-2266 25 mg and mannitol 60 mg added 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 at least 6 months at 2 to 8°C.

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

Chemistry

Chemical name: H-D-Valyl-L-leucyl-L-argininep-Nitroaniline dihydrochloride

Formula: H-D-Val-Leu-Arg-pNA · 2HCl

S-2266 structure

Mol. wt: 579.6

e316 nm: 1.27 · 104 mol-1 · l · cm-1

Solubility: > 40 mmol/l in H2O

Suitable stock solution: 2 mmol/l in H2O

Principle

	Enzyme
H-D-Val-Leu-Arg-pNA		H-D-Val-Leu-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.

Kinetic Data

Porcine pancreas kallikrein: Km=2.2 · 10-5 mol/l, V=8 · 10-9 mol/min · KU
Human urine kallikrein: Km=3 · 10-5 mol/l
Human saliva kallikrein: Km=5 · 10-4 mol/l. Determined at 37°C in 2.5 ml of 0.05 mol/l Tris buffer pH 9.0, I 0.05.

Standardization

An activity of DA/min= 0.05 (37°C) is obtained by using 0.1 mmol/l of the substrate and 0.6 KU/ml of porcine pancreas kallikrein, KZC (Bayer, Leverkusen, Germany).