Caspases,Paracaspases, and Metacaspases Methods and Protocols

33

used for linear regression result in a good correlation factor

(r^2 > 0.98). Most caspase preparations will have a titer that is

slightly below (75–95 %) the protein concentration estimated

using the Edelhoch method. A titer that is higher often reveals

inefficient inhibition by Z-VAD-fmk. In this case, inhibition

can be prolonged for 1 h, and/or caspase and Z-VAD-fmk

concentrations can be proportionally increased. Nevertheless,

Z-VAD-fmk may not be potent enough to react effectively

with some caspase mutants. In this case, we recommend titra-

tion using the baculoviral protein p35. This pan-caspase inhib-

itor is much more potent at inhibiting caspases [ 49 , 50 ]. p35

is expressed as a C-terminal His-tagged protein and purified

using the same procedure as the one described for caspase-3

(Subheading 3.1.1). However, because it is a protein, p35

must be titrated before using it as a titrant. A titration reaction

is set up with a wild-type caspase-3 preparation that has already

been titrated using Z-VAD-fmk. In this case, the x-axis inter-

cept (y = 0) corresponds to the concentration of p35 that is

Table 2
Caspase internal cleavage site and specificity

Molecular form

expressed

Internal cleavage site

(P 4 P 3 P 2 P 1 )a

Substrate

preference (P 4 P 3 P 2 P 1 )

Synthetic substrate

used (P 4 P 3 P 2 P 1 )

Caspase-2 DQQD (linker) (V)DEHD VDVADb
Caspase-3 ESMD (N-terminal domain) DEVD DEVD
IETD (linker)
Caspase-6 DVVD (linker) VEHD VEID
TEVD (linker) DEVD
TETD (N-terminal domain)
Caspase-7 DSVD (N-terminal domain) DEVD DEVD
IQAD (linker)
NDTD (linker)
Caspase-8 VETD (linker) (I/L)ETD IETD
LEMD (linker) VEID
REQD (N-terminal domain) DEVD
Caspase-9 PEPD (linker) (I/L)EHD LEHD
DQLD (linker) IETD
Caspase-10 IEAD (linker) LEHD LEHD
SQTD (N-terminal domain) DEVD
aInternal cleavage sites occurring between the N-terminal domain and the catalytic site or within the linker that separates
the large and small subunit of the catalytic domain are indicated. All those cleavages can be observed during overexpres-
sion in E. coli, but not always during apoptosis
bThe most complete studies used a tetrapeptide library to establish caspase-2 peptidic substrate preference [ 4 ]. However,
others have shown that caspase-2 has an extended substrate-binding pocket, which explains why this caspase is more
active on pentapeptides [ 25 ]

Apoptotic Caspases Assays