35
If KM is too high (>200 μM), it is better to determine the
kcat/KM value using pseudo-first order conditions. If [S] ≪KM,
Eq. 2 can be simplified to v ≈ Vmax[S]/KM, which can be rear-
ranged to Vmax/KM ≈ v/[S]. If pseudo-first order conditions are
met, a plot of rates against substrate concentrations gives a
straight line with a slope equivalent to Vmax/KM, and
kcat/KM = Vmax/KM[E]. As explained in the introduction of this
section, kcat/KM is a useful value to compare enzymes. Because
the obtained value is an approximation, it is more appropriate
to refer to an apparent value, such as kcat,app/KM,app. If one can-
not assume that pseudo- first order conditions are met, the
value can be referred to as a rate constant.
- An alternative protocol is to employ the mRIPA lysis buffer
(50 mM Tris at pH 7.4, 100 mM NaCl, 1 % NP-40, 0.5 %
deoxycholic acid, 0.1 % sodium dodecylsulfate (SDS), and
1 mM EDTA). This solution extracts more proteins, but not
only cytosolic proteins.
- Use initiator caspase buffer if working with initiator caspases,
and incubate a 1 μM initiator caspase solution for 30 min at
37 °C before setting up the serial dilution. To prepare the 2/3
serial dilution, 25 μL of 1× caspase buffer is added to tubes
#2–8, 75 μL of 200 nM of caspase is added to tube #1, and
50 μL is transferred from tube to tube (tubes #1–8) by repeat-
edly pipetting up and down to mix.
- Initiator caspase buffer may cause the precipitation of lysate
protein or recombinant proteins. It is recommended to test the
solubility of the substrate in buffer before performing the assay.
If precipitation occurs, centrifuge the tube and test the super-
natant for the protein of interest. Diluting the substrate/lysate
will reduce precipitation and help meet the pseudo- first order
condition.
- A more accurate estimation of k is obtained by calculating the
value for all samples and averaging them. The values obtained
should be roughly similar for a pseudo-first order condition.
Alternatively, plot ln(1−p)/t against [E] for all samples. The
slope will be −k.
- Because the efficacy of cleavage may vary greatly, it is likely that
results will show full cleavage of the protein of interest or
barely any cleavage. This situation is why it is suggested to start
with a caspase concentration that is relatively high, yet physi-
ological. The experiment is then repeated using a different and
narrower range of caspase concentrations and a different reac-
tion time to more accurately determine kcat/KM. Whenever fea-
sible, do not use concentrations of caspase that are too high
(>100 nM), and lower the amount of substrates in the assay.
The lower the substrate concentration is, the more likely the
reaction will occur in a pseudo-first order condition. If samples
Apoptotic Caspases Assays
