Caspases,Paracaspases, and Metacaspases Methods and Protocols

16

and techniques are mastered, the full characterization (titration

and kinetic parameter determination) can be accomplished in less

than a day.

The determination of enzyme velocity requires that experimental

values be represented in molar amount of product generated per

unit of time (e.g., μM/s or nM/s). Therefore, the relationship

between relative fluorescence units (RFU) given as readouts from

the plate reader and the actual concentration of product must be

determined. The conversion factor varies greatly between instru-

ments and system settings for the same instrument. It also varies

over time for the same instrument and settings. For instance,

instruments that use a lamp for excitation will, over the lifetime of

the lamp, display a decreased performance. Therefore, an accurate

velocity measurement requires frequent determination of the rela-

tionship between RFU and the corresponding molar amount of

product. To this end, one must first make a standard fluorophore

solution. This solution is used to calibrate the spectrofluorometer.

In a similar fashion to the Afc standard, the substrate must also

be calibrated. Indeed, the purity of commercially available fluoro-

genic substrates varies among providers and from lot to lot within the

same manufacturer. A simple way to obtain accurate and reproduc-

ible values using commercial substrates is to calibrate them following

the complete hydrolysis of a fixed amount of substrate. This process

ensures that the concentration of “usable” substrate is determined.

To produce reliable and reproducible data, important technical

considerations must be taken into account. First, it is critical to

use the same brand and model of plates for calibration of the plate

reader and enzymatic assays. This requirement arises because the

plate used significantly influences the relationship between the flu-

orescence readout and the actual concentration of the fluorophore.

It is also critical that the instrument settings used to calibrate the

plate reader are exactly the same as the ones used for all enzymatic

assays. Otherwise, the conversion of fluorescence readout into

molar concentration of product will no longer stand. Consequently,

it is recommended to generate several datasets of calibration for

every group of settings that will be used. If available, softwares or

instrument programs, templates or methods are useful to save the

instrument parameters.

This procedure takes 1 h to be completed.

1. Weigh a small amount of Afc and prepare a 10 mM solution in
   DMSO.


2. Using the stock solution, prepare 1 mL of 50 μM Afc solution
   in water.


3. Measure the absorbance of the diluted Afc solution at 380 nm
   in a 1 cm path quartz cuvette. Determine the actual concentra-
   tion of Afc using the molar extinction coefficient (ε) of Afc

3.2.1 Substrate and
Plate Reader Calibration

Standard Afc Solution

Dave Boucher et al.

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