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small subunit (~10–12 kDa). The generation of those subunits
is the result of the auto-catalytic activation of caspases during
overexpression in E. coli but does necessarily reflect the normal
activation mechanism of caspases in eukaryotic cells. With some
caspase mutants, this process may not be complete. Usually,
increasing the expression time will resolve this problem.
- The Edelhoch relationship uses the observed absorbance of
the tryptophan, tyrosine and reduced/oxidized cysteine resi-
due content of a protein as a basis for calculating the extinction
coefficient [ 48 ]. The ProtParam algorithm provides the esti-
mated extinction coefficient and is available online at http://
http://www.expasy.org. The value obtained is used to calculate the
initial enzyme concentration for active site titration.
- Do not refreeze caspase aliquots. Like many enzymes, caspases
do not appreciate freeze–thaw cycles. The caspase preparation
will be stable for months at −80 °C. However, caspase-6 has a
shorter shelf life and should be active site-titrated every time.
We highly suggest performing active site titration every month
or prior to use.
- From this step and until further notice, it is important to pro-
ceed at room temperature to avoid guanidine and urea crystal-
lization and full-length caspase-8 precipitation.
- Caspase-8 prepared using the above protocol is unstable and
must be analyzed immediately. The more quickly this analysis
is performed, the higher the active site titer of the preparation
will be; this will also be reflected in the kinetic parameter, kcat.
- Because this text is oversimplified, it is strongly recommended
that readers consult enzymology textbooks for a more thor-
ough discussion of enzyme kinetics.
- As an alternative to Afc fluorophores, 7-amino-4-
methylcoumarin (Amc; ε354 nm = 17,800 M−1 cm−1; EXλ = 380 nm
and EMλ = 460 nm)-based fluorogenic substrates can be used.
Amc fluorophore has a smaller Stokes’ shift (separation between
excitation and emission spectra) upon cleavage than Afc.
Chromogenic substrates based on p-nitroaniline (pNA;
ε410 nm = 8,800 M−1 cm−1) could be used, but because their
dynamic range of absorbance is much smaller than that of most
fluorophores, pNA-based substrates are less useful. Prepare
Amc and pNA standard solutions in DMSO.
- The buffer used will alter the emission and excitation intensi-
ties, a phenomenon called quenching. Therefore, the plate
reader must be calibrated for every buffer (e.g., executioner
vs. initiator caspase buffer). It is also important that the vol-
ume of the sample is exactly the same as the one used in
enzymatic assays.
Apoptotic Caspases Assays
