Caspases,Paracaspases, and Metacaspases Methods and Protocols

19

Executioner caspases (caspase-3, caspase-6, and caspase-7) are

fully active and dimeric in a buffer that closely mimics the cytosol

(e.g., executioner caspase buffer). On the contrary, initiator cas-

pases are activated by dimerization on multimeric platforms such as

the DISC (death-inducing signaling complex) for caspase-8 and

caspase-10, the apoptosome for caspase-9, and the PIDDosome

for caspase-2 [ 26 ]. These complexes recruit initiator caspases via

homotypic interaction of death domain superfamily domains:

caspase- 8 and caspase-10 use tandem death effector domains

(DEDs), and caspase-2 and caspase-9 employ a single caspase acti-

vation recruitment domain (CARD) [ 26 ]. Because initiator cas-

pases are usually purified as a mixture of dimers (active) and

0.0

0.2

0.4

0.6

0.8

1.0

ab

cd

0.0 0.2 0.4 0.6 0.8 1.0

Z-VAD-fmk (μM)

y = -18.26x + 0.88

r^2 = 0.99

0.0

0.2

0.4

0.6

0.8

1.0

0.00 0.02 0.04 0.06

0.0

0.2

0.4

0.6

0.8

1.0

0.00.2 0.40.6 0. 81 .0

Relative fluorescence

(RFU/min)

Z-VAD-fmk (μM)

y = -14.22x + 0.83

r^2 = 0.94

0.0

0.2

0.4

0.6

0.8

1.0

0.000.020.040.060.08

0.0

0.2

0.4

0.6

0.8

1.0

0.0 0.2 0.4 0.6 0.8 1.0

Relative fluorescence

(RFU/min)

Relative fluorescence

(RFU/min)

Z-VAD-fmk (μM)

0.0

0.1

0.2

0.3

0.4

0.5

0.0 0.2 0.4

Calculation

Using data in A inset:

x value (Z-VAD-fmk concentration)

at y = 0 (100% inhibition)

is 0.048 μM (48 nM), i.e., x = -0.88/-18.26

Because 100 nM were used in the titration assay, the

preparation is 48% active

Caspase concentration estimated (Edelhoch method)

at 15.2 μM

Actual caspase preparation contains 7.3 μM active

sites (15.2 μM x 48% = 7.3 μM)

Fig. 4 Caspase titration. (a) Example of a good titration dataset of caspase-7 and good analysis of titration
data. Data points used (gray area) to determine the titer are appropriately chosen resulting in an accurate titer
determination. The data used follow the linear regression well. (b) The same titration dataset as in (a) but with
poor analysis of titration data. Data points used (gray area) to determine the titer were poorly chosen, resulting
in overestimation of the titer. The inset shows that the value at 0.06 μM Z-VAD-fmk clearly departed to the right
of the linear regression set by all values, which suggests that inhibition was not complete in this sample.
(c) Example of a titration experiment with incomplete inhibition leading to an inappropriate titration. The inset
shows data describing a curve instead of a straight line. In this case, the titration should be repeated.
(d) Calculation for caspase-7 titration using data in (a)

Apoptotic Caspases Assays