SPECTROZYME® FVIIa, chromogenic substrate for FVIIa assays

Description
Chromogenic substrate for amidolytic assay of Factor VIIa and lipidated Tissue Factor/VIIa complex.

Chemistry
Formula: CH₃SO₂-D-CHA-But-Arg-pNA.AcOH
Chemical name: Methanesulphonyl-D-cyclohexyl-alanyl-butyl-arginine-paranitroaniline monoacetate salt
MW: 670.8 Dalton
Solubility: > 10 mM in distilled/deionized water,
                  > 0.5 mM in Tris Buffer, pH 8.4

Suggested Use
SPECTROZYME^® FVIIa, it is the substrate of choice in all chromogenic based methods for the determination of factor VIIa, and for the factor VIIa/Tissue Factor complex in particular.

Assay Conditions/SubstrateKinetics
Enzyme activity is determined by measuring the increase in absorbance of the free chromophore (pNA) generated, in comparison to the original substrate, per unit time at λ405 nm. At excess substrate concentration, the rate of absorbance increase due to the amount of chromophore released is linearly related to enzyme concentration. Measurement can be made either through acid quenching of the reaction (end-point method), or through use of a kinetic recording spectrophotometer (initial-rate-method).

Kinetic Data for Factor VIIa and TF complex
Buffer:  880 µL Tris-Buffer, pH 8.4       Temp. 37°C
            (0.1M Tris, 0.1M NaCl, 5 mM CaCl₂, 0.1% BSA)               
Substrate: 0.100 mL of No. ADG217 at 4 mM
Enzyme:
- human factor VIIa
  0.020 mL of FVIIa at 0.025 mg/mL
 => K_M : 5.0 mM       V_max: 6.72 nmoles/min

- By adding Tissue Factor with a 10:1 ratio (TF:FVIIa)
   0.100 mL of FVIIa at 0.025 mg/mL  +
     0.100 mL of tissue factor at 0.25 mg/mL
      => K_M : 0.97 mM       V_max: 69.7 nmoles/min
 
Presentation
Glass vial containing 50 μmoles of lyophilized substrate.

Reconstitution
Reconstitute with filtered distilled/deionized water to create a stock solution with a concentration of 5.0 mM. The typical working range concentration is 4.0 mM.

Storage
Store intact vials in the dark at 2 - 8 °C for up to 5 years from the date of manufacture. Reconsituted substrate may be stored for 1 week at room temperature, 2 months at 2° - 8°C, or for up to 6 months at –20°C (Aliquot and freeze. Do not submit to freeze-thaw cycles).

Category: Research use only

Type: Substrate

Product Availability: Worldwide

Manufacturer: ImmBioMed GmbH & Co KG, Germany

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