Description

Highly sensitive chromogenic substrate for factor

IXa. The sensitivity of this substrate is significantly

enhanced in the presence of alcohols, especially

ethylene glycol.

Chemistry

Formula: CH3SO2-(D)-CHG-Gly-Arg-pNA·AcOH

Chemical name: Methylsulfonyl-D-cyclohexylglycylglycyl-

arginine-paranitroanilide monoacetate salt

MW: 628.7 Dalton

Solubility: up to 20 mM in water

Biochemical Characteristics:

kcat = 4.4/sec KM = 1.3 mM

kcat/KM= 3.38 l/mM·s (determined in the presence

of 33% ethylene glycol)

Intendet use

Determination of Factor IXa in concentrates or other

blood products according to Ref 1.

Conditions: The test temperature can be selected

but should be kept constant during the assay. All

reagents should be kept at the test temperature prior

to use. Do not work with chilled reagents directly

from the refrigerator. For the kinetic version 37°C

may be used, especially when a thermo-stated cell

holder is available.

Automation: The assay can be either performed on

a spectrophotometer or microtiter plate reader at 405

nm. Kinetic or endpoint versions are possible. An

adaptation on fully automated chemistry analyzers at

405 nm may be possible but has not been tested.

Buffer: 50 mM Tris, pH 7.4, 100 mM NaCl, 5 mM

CaCl2, 40 % (vol/ vol) ethylene glycol.

Notes: The sensitivity of this substrate for factor IXa

is significantly increased in the presence of 33%

ethylene glycol.

All volumes of the described pipetting scheme may

be adapted for assay in regular cuvettes. An

example is given below.

Interference by turbidity or from colored samples in

the endpoint assay can lead to falsely elevated

results. This can be prevented by running a sample

blank as follows:

Pipette a sample blank in the following sequence:

Acetic Acid/„Stop reagent“ - Buffer sample -

Substrate

Microtiter plate format

0.200 ml Buffer

0.025 ml Spectrozyme® FIXa (10 mM)

0.020 ml Sample (factor IXaβ, 2 μM)

⇒ Determination of optical density at 405 nm

0.025 ml Acetic acid (50 %) to stop the reaction after

5-10 minutes

Spectrophotometer format

0.800 ml Buffer

0.100 ml Spectrozyme® FIXa (10 mM)

0.080 ml Sample (factor IXaβ, 2 μM)

⇒ Determination of optical density at 405 nm

0.100 ml Acetic acid (50 %) to stop the reaction after

5-10 minutes

Calculate the activity of factor IXa according to:

F IXa activity = (OD sample – OD sample blank)

Presentation

ADG299 - amber glass vial containing 10 μmoles of

substrate.

ADG299B – amber glass vial containing 0.5 grams

(approx. 795 μmoles) of substrate.

Reconstitution

Reconstitute with filtered deionized water to create

a 10 mM solution. Shake gently before use.

Storage

May be used by the expiry date given on the label

when stored unopened, protected from moisture, in

the dark, 2-8°C. Avoid contamination of the reagents

by microorganisms.

Shipment of product does not require cooling during

the time of transportation.

References

1. Dramatic enhancement of the catalytic activity of

coagulation factor IXa by alcohols. Stürzebecher

J, Kopetzki E, Bode W, Hopfner KP. FEBS Lett

1997; 412:295-300

2. Determination of activated factor IX in factor IX

concentrates with a chromogenic substrate.

Prasa D, Stuerzebecher J. Throm Res; 92:99-

102