Substrates containing the fluorescent reporter group 6-amino-1-naphthalene-sulfonamide (ANSN) are useful compounds for monitoring the enzyme activity of various serine proteases (1-12). In this class of compounds, the ANSN reporter group is linked (in the R1 position) to short tri-peptide sequences. The peptide sequences are designed to optimize the interaction between the enzyme and substrate. Additional components which may be added to the R2 and R3 positions reflect changes in the P' subsite positions, and generally affect the kinetic parameters of the substrates. Compounds which are effective substrates are hydrolyzed between the tri-peptide and the ANSN group. Once cleaved from the peptide moiety, the ANSN group exhibits about a 1000 fold increase in relative fluorescence.

The kinetic properties listed below will aid in the selection of an appropriate substrate. The ANSN substrates have proved especially useful for the analyses of factor VIIa (1-5,7,9,10,12). Although the substrate hydrolysis rates are relatively slow for factor VIIa alone, several compounds exhibit a large change in kcat when tissue factor is incorporated into the assay system.

Form:
Supplied as a liquid in DMSO.

Assay Physiologic Buffers:
Hepes or Tris with substrate concentrations ranging from 1-100uM

Emission Wavelength:
470nm

Excitation Wavelength:
352nm

Light Artifacts:
Minimized by using a 390-450nm long-pass cutoff filter in the emission beam.

Storage and Stability:
May be stored at 4°C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20°C. Aliquots are stable for 6 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Light sensitive.

Note:
DMSO may be a solid (frozen) at 4°C. Allow product to come to room temperature prior to use.