Choice of Fluorophore

The selection of fluorophore is made so that both excitation and emission are in the visible wavelength region. The rationale for this specific dye selection is that biological materials contain molecules that both absorb and fluoresce in the UV wavelength domain; this autofluorescence can hinder determination of small amounts of activity present in samples and reduce the reliability of the activity determination.

Substrate Design and Computer Modeled Structure

Peptide substrates are synthesized with covalently coupled fluorophores. The latter are strategically situated away from the target sequence to ensure noninterference with proteolytic cleavage.

A representative computer modeled structure for elastase substrate is shown below where the yellow ribbon represents the peptide backbone conformation and the cyan color the attached fluorophores. Green arrow indicates the cleavage site.

Two different views of this peptide are shown.

 Note: First, the peptide backbone assumes a loop conformation similar to
 that found in reactive site loops of SERPINs.

 Second, the cyan colored dye moieties form a dimer. This further stabilizes
 the peptide backbone conformation. This molecular configuration can be best
 described by the formation of a nonfluorescent ground-state dimer.

Cleavage-induced Fluorescence and Absorption changes

In the intact peptide the cyan colored fluorophores form a ground-state dimer. The absorption spectrum of this ground-state dimer can best be described by exciton theory. Peptide cleavage abolishes this dye-dye interaction and results in an increase in fluorescence and significant absorption changes.

References

On Exciton theory used in fluorogenic protease substrate design:

1. B. Z. Packard, D. D. Toptygin, A. Komoriya, and L. Brand Profluorescenct protease substrates: intramolcular dimers described by the exciton model. Proc. Natl. Acad. Sci. (USA) 93: 11640-11645 (1996).

2. B. Z. Packard, D. D. Toptygin, A. Komoriya, and L. Brand. The design of fluorogenic protease substrates guided by exciton theory. Meth. Enzym. 278: 15-28 (1997).

3. B. Z.Packard, A. Komoriya, D. D.Toptygin, and L. Brand. Structural characteristics of fluorophores which form intramolecular H-type dimers in a protease substrate. J. Phys. Chem. B 101: 5070-5074 (1997).

4. B. Z. Packard, D. D. Toptygin, A. Komoriya, and L. Brand. Characterization of fluorescence quenching in bifluorophoric protease substrates. Biophys. Chem. 67: 167-176 (1997)

5. B. Z. Packard, D. D. Toptygin, A. Komoriya, and L. Brand. Intramolecular resonance dipole-dipole interactions in a protease substrate. J. Phys. Chem. B 102: 752-758 (1998).

6. B. Z. Packard, A. Komoriya, V. Nanda, and L. Brand. Intramolecular excitonic dimers in protease substrates: modification of the backbone moiety to probe the H-dimer structure. J. Phys. Chem. B 102: 1820-1827 (1998).

On Caspase-3 nomenclature:

7. E.S. Alnemri et al. Human ICE/CED-3 Protease Nomenclature. Cell 87: 171 (1996)

On DEVD-based substrate and inhibitor characteristics:

8. A. Sarin, M.-L. Wu, and P. A. Henkart. Different Interleukin-1b Converting Enzyme (ICE) family protease requirements for the apoptotic death of T-lymphocytes triggered by diverse stimuli. J. Exp. Med. 184: 2445-2450 (1996)

9. D. W. Nicholson et. al. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 376: 37-43 (1995).

Intracellular Caspase-3-like activities determination using PhiPhiLux:

10. H. Hirata, A. Takahashi, S. Kobayashi, S. Yonehara, H. Sawai, T. Okasaki, K. Yamamoto, and M. Sasada. Caspases are activated in a branched protease cascade and control distinct downstream processes in Fas-induced apoptosis. J. Exp. Med. 187: 587-600 (1998)

11. J. M. Zapata, R. Takahashi, G.S.Salvesen and J.C. Reed. Granzyme release and caspase activation in activated human T-lymphocytes.  J. Biol. Chem. 273: 6916-6920 (1998).

12. R.M. Siegel, D.A. Martin, L.Zheng, S.Y. Ng, J. Cohen, and M.J. Lenardo. Death-effector filaments: novel cytoplasmic structures that recruite caspases and trigger apoptosis. J. Cell Biol. 141: 1243-1253 (1998).

13. L. Guedez, W.G. Stetler-Stevenson, L. Wolff, J. Wang, P. Fukushima, A. Mansoor, M. Stetler-Stevenson. In vitro suppression of programmed cell death of B cells by tissue inhibitor of metalloproteinases-1. J. Clin. Invest. 102: 2002-10 (1998).

14. A. Komoriya, B.Z. Packard, M.J. Brown, M.-L. Wu, and P.A. Henkart. A novel intracellular caspase fluorogenic substrate for the early detection of apoptosis in thymocytes. (in preparation).