Thrombin Substrate (Boc-L-FPR-ANSN-H-C2H5)

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.\n\nThe 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.\n\nForm:\nSupplied as a liquid in DMSO.\n\nConcentration:\n7.5mg/ml\n\nAssays physiologic buffers:\nHepes or Tris, with substrate concentrations ranging from 1-100uM\n\nEmission Wavelength:\n470nm\n\nExcitation Wavelength:\n352nm\n\nLight artifacts:\nMinimized by using a 390-450nm long-pass cutoff filter in the emission beam.\n\nStorage and Stability:\nMay be stored at 4 degrees C for short-term only. For long-term storage, store at -20 degrees C. Aliquots are stable for at least 6 months at -20 degrees C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.

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SPECIFICATIONS

Catalog Number

T5045-11

Size

1mg

References

1. Butenas, S., et al., Biochemistry 31: 5399 (1992). 2. Lawson, J.H., et al., J. Biol. Chem. 267: 4834 (1992). 3. Lawson, J.H., et al., Methods in Enzymology 222: 177 (1993). 4. Butenas, S., et al., Biochemistry 32: 6531 (1993). 5. Butenas, S., et al., J. Biol. Chem. 269: 25,838 (1994). 6. Butenas, S., et al., Anal. Biochem. 225: 231 (1995). 7. Butenas, S., and Mann, K.G., Biochemistry 35: 1904 (1996). 8. Butenas, S., et al., Biochemistry 36: 2123 (1997). 9. Butenas, S., et al., J. Biol. Chem. 272: 21,527 (1997). 10. Butenas, S., et al., Thromb. Haemost. 78: 1193 (1997). 11. Hockin, M.F., et al., Arterioscler. Thromb. Vasc. Biol. In press (1997). 12. Butenas, S., et al., U.S.A. patent # 5,399,487.