Caspases,Paracaspases, and Metacaspases Methods and Protocols

17

(ε 380 nm = 12,600 M−1 cm−1). A 50 μM solution of pure Afc has

an absorbance of 0.630 at 380 nm (see Note 19).

4. Correct the initial concentration of Afc accordingly. This is the
   standard Afc solution. Afc solution is stable for at least 1 year at
   −20 °C.

This procedure takes 1 h to be completed.

1. Using the standard solution of fluorophore (step 4,
   Subheading 3.2.1.1), set up a series of 100 μL Afc samples in a
   microplate in 1× caspase buffer to cover a concentration range
   between 1 and 100 μM (15 samples). Plan more samples in the
   1–10 μM range than in the higher range. Include a buffer-only
   sample (see Note 20).


2. Read the fluorescence at EXλ = 405 nm and EMλ = 510 nm for
   every setting of the instrument that will be used (e.g., various
   values of gain, wavelength bandwidth, flashes, integration time).


3. Plot the fluorescence of each sample against the Afc concentra-
   tion and determine the slope of the straight portion of the
   trace by linear regression. The slope describes the relationship
   between RFU and Afc concentration.


4. Repeat the procedure regularly (see Note 21).

This procedure takes 2 h to be completed.

1. Based on the amount indicated on the product label, dissolve
   a vial of substrate in DMSO to obtain a final concentration of
   30 mM.


2. Set up a 1 mL caspase reaction containing 50 μM Afc-based
   substrate in 1× executioner caspase buffer and add excess puri-
   fied recombinant caspase-3 (100 nM) to completely hydrolyze
   the available substrate. Set up another sample without caspase.
   Incubate for 1 h at 37 °C in the dark (see Note 22).


3. Measure the absorbance of the reaction at 380 nm in a 1 cm
   path quartz cuvette. Use the caspase-free sample as a blank.
   Determine the actual concentration of “usable” substrate
   using the molar extinction coefficient (ε) of Afc
   (ε380 nm = 12,600 M−1 cm−1) (see Notes 19 and 23 ).


4. Correct the initial concentration of Afc-based substrate accord-
   ingly. Fluorogenic substrate solutions are stable for at least
   1 year at −20 °C.

The goal of active site titration is to determine the molar amount

of active sites in an enzyme preparation. Too often, enzymes (even

caspases) are provided as unit amount, for example, restriction

enzymes used in molecular cloning. Although this approach is sat-

isfactory because enzymes are often used to perform a certain

quantity of work (e.g., cleaving 10 μg of plasmid DNA) and

Plate Reader Calibration

Fluorogenic Substrate
Calibration

3.2.2 Caspase Active
Site Titration

Apoptotic Caspases Assays