21- Extract the initial rate (straight portion of the fluorescence
plotted against time) of every reaction and plot them against
Z-VAD-fmk concentration (step 3). Utilize linear regression to
obtain the x-axis intercept (y = 0) using data starting from the lowest
Z-VAD- fmk concentration up to the value at which ~10 % of
enzyme activity remains. The intercept is equal to the concentra-
tion of caspase in the titration reaction (step 3) (see Note 28). - Because the estimated concentration of caspase in the titration
reaction was set at 100 nM based on protein quantity, correct
the value accordingly.
Practically, a series of enzymatic reactions are set up in a micro-well
plate with a serial dilution of the caspase substrate. The hydrolysis
of that substrate is measured continuously in a plate reader. For
most caspases, the hydrolysis rate will follow the typical Michaelis–
Menten equation. The initial velocity of each sample is used to
determine the kinetic parameters using nonlinear regression. The
protocol describes how to determine the kinetic parameters for an
Afc fluorogenic substrate. However, the same protocol can be used
for other type of substrates, including chromogenic substrates. In
the latter case, a spectrophotometer is used, and a standard curve
of the free chromophore is used to convert the absorbance into
molar amount of product generated.
See protocol in Subheading 3.2.2 and accompanying notes.
This procedure takes 2 h to be completed.
Protocol- Thaw an aliquot of active site-titrated caspase on ice (this step takes
approximately 30 min). Do not heat the sample (see Note 29). - In a 96-well plate, set up a 3/4 serial dilution of Afc fluorogenic
substrate in 1× caspase buffer (50 μL/well, starting at 300 μM
or higher if necessary) over 16 wells (see Notes 30 and 31 ). - For initiator caspases only: prepare 100 μL of 1 μM caspase
solution in 1× initiator caspase buffer. Incubate at 37 °C for
30 min. This solution is used to prepare the diluted caspase
solution in step 4. - Prepare 1 mL of a twofold concentrated caspase solution (i.e.,
2–40 nM) in 1× caspase buffer. With a repeating pipettor, rap-
idly add 50 μL of this solution to every well of the plate.
Thoroughly mix and immediately read the fluorescence at
EXλ = 405 nm and EMλ = 510 nm for 30 min at 37 °C. Ideally,
take measurements every 5 s. - Extract the initial rate of every reaction, convert the rates to
μM/min or nM/s using the RFU to μM/nM relationship
(Subheading 3.2.1.2) for the specific instrument settings used,
and plot rates against substrate concentration. Use nonlinear
regression and Eq. 2 to extract Vmax and KM. Calculate kcat
using kcat = Vmax/[E] (see Note 32).
3.2.3 General Protocol to
Determine KM and kcat of a
Fluorogenic Substrate
Apoptotic Caspases Assays