34
required to completely inhibit caspase-3. For example, if the
titration was performed using 100 nM caspase-3, an intercept
at 500 nM means that only 20 % of the p35 preparation is
active, which is a reasonable value. Thus, the value of active
p35 is 20 % of the concentration that has been determined
using the Edelhoch method.- Because enzyme addition to the reaction takes some time, more
reliable data are obtained if no more than two series of assays are
performed simultaneously. However, one can determine the
kinetic parameters for multiple substrates simultaneously if an
automatic injector is used to add the enzyme and reading can be
programmed to alternate injection and reading. - To set up the 3/4 serial dilution: add 50 μL of 1× caspase buf-
fer to wells #2–16, add 200 μL of 600 μM substrate to well #1,
and transfer 150 μL from well to well (wells #1–16) by repeat-
edly pipetting up and down to mix at every transfer. - Make sure that the final DMSO concentration is not >3 %. DMSO
will negatively affect caspase activity at high substrate concentra-
tion and consequently artificially alter KM and kcat values. - It is important that the data used for linear regression result in a
very good correlation factor (r^2 > 0.98). Otherwise, this may
indicate that mistakes have been made during the experiments
or that the enzyme does not follow the classical Michaelis–
Menten mechanism, for example through enzyme cooperativity,
product inhibition, inhibition by DMSO. The latter often occurs
for the 300 μM substrate sample. If this is the case, this data
point can be omitted. Care must be taken to select the time
interval used for parameter determination. Do not take all mea-
surements, but only an early interval during which the data show
constant rates (2–10 min). This instruction is given because later
time point values underestimate caspase activity due to substrate
depletion or enzyme fatigue (e.g., caspase-6).
Make sure that the enzyme concentration, rates, and RFU to
product concentration conversion number are in the same
molar unit. For example, if [E] is in nanomolar, convert rates
to nM/s and RFU numbers to nM. Remember that kcat is a
number of molecules per second and not per minute.
If KM is high (>100 μM), the above procedure will result in less
accurate KM and kcat values. This phenomenon arises because
not enough assays are above KM. Ideally, half the data points
should be above KM, and the best estimates are obtained with
data that populate the inflection point of the Michaelis–Menten
curve, between 0.1 and 10 times KM. If KM is too high or too
low, it is recommended to repeat the experiment using a differ-
ent range of substrate concentrations. Accordingly, it may be
necessary to prepare substrate stocks that are more concen-
trated (e.g., 50 mM instead of 20 mM).
Dave Boucher et al.http://www.ebook3000.com