Caspases,Paracaspases, and Metacaspases Methods and Protocols

245

Although different metacaspases have many biochemical features

in common (e.g., active as monomers and Ca 2+ -dependent), the

optimal pH range for catalysis varies signifi cantly necessitating

optimization of conditions for each individual metacaspase. We

have optimized activity assay for recombinant 6× His-tagged mcII-

Pa using AMC-labeled fl uorogenic substrates FESR-AMC

(custom- synthesized; matches mcII-Pa autoprocessing site), EGR-

AMC (e.g., from Bachem), or Ac-VRPR-AMC (Bachem I-1965).

AMC is a fl uorescent compound, which is highly stable in aqueous

solutions and changes its maxima of excitation/emission after

being released from a peptide upon carboxypeptidase activity [ 19 ,

20 ]. Detection of free AMC fl uorescence gives a high precision

and sensitivity readout of proteolytic activity in the reaction.

1. Keep all samples on ice before measurement.


2. Dilute substrate with reaction buffer to a fi nal concentration
   of 1 mM.


3. Prepare a blank sample by mixing 152 μL of reaction buffer
   with 8 μL of 1 mM substrate.


4. Prepare fi ve standard samples by making serial dilutions of
   24 μM AMC stock using reaction buffer to obtain 12, 6, 3,
   1.5, and 0.75 μM.


5. Pipet 50 μL of each standard solution and the blank solution
   into 96-well opaque plate in triplicates.


6. Prepare two dilutions of recombinant metacaspase purifi ed in
   native conditions ( see Subheading 3.1 ) with reaction buffer con-
   taining 50 mM CaCl 2 and without calcium. Final concentration
   of the diluted protein should be 80 μg/mL ( see Note 6 ).


7. Pipet 47.5 μL of the diluted metacaspase into 96-well opaque
   plate (3 ng of metacaspase per reaction) in triplicates.


8. Add 2.5 μl of 1 mM substrate to each well with diluted meta-
   caspase (fi nal concentration of substrate 50 μM).


9. Set the detector gain to 70 % using the 3-μM AMC standard.


10. Set the temperature in the plate reader chamber in the range
    25–28 °C.


11. Shake the plate in the plate reader for 10 s and take reads of
    fl uorescence intensity at excitation/emission 360–380 nm/
    440–460 nm every min making at least 20 fl ashes per well.
    Take reads for at least 15 min.


12. Use the standard curve and linear part of the reaction curve to
    calculate pmol of AMC released per minute per nanogram of
    metacaspase.


13. Subtract values obtained for samples without calcium from the
    values of samples containing calcium ( see Note 6 ).

3.4 Measurement
of Recombinant
Metacaspase Activity

Plant Metacaspases