215- Read the fl uorescence using an excitation wavelength of
355 nm and emission wavelength of 460 nm. Fluorescence
measurements should be taken approximately every 5 s for
around 10 min, but the interval time and total read time can
be varied, depending on the reaction velocity. - Plot the relative fl uorescence units (FU) versus time ( T ) (in min).
- Select a time interval over which the increase in fl uorescence is
linear and calculate the slope of the graph over this time frame.
This gives the reaction rate ( Δ FU/ T ), which is a relative mea-
sure of protein activity. This can generally be used to compare
the activities of different enzymes within the same experiment
(on the same 96-well plate).
In order to calculate the specifi c activity of the protein an AMC
standard curve should be generated.- Make a 10 μM solution of AMC in ddH 2 O (AMC 1) in a
microcentrifuge tube. - Make a 1/2 (2-fold) dilution of AMC 1 with ddH 2 O (e.g.,
200 μL AMC + 200 μL ddH 2 O) and place in tube 2. - Repeat this 2-fold serial dilution in to further nine tubes.
- Add 200 μL of each dilution to a single row (wells 1–11) of a
96-well plate and add 200 μL ddH 2 O to well 12. - Remove the plate from the ice, place in the fl uorescence plate
reader, and equilibrate to ambient temperature (20–25 °C) for
5 min. - Read the fl uorescence using an excitation wavelength of
355 nm and emission wavelength of 460 nm ( see Note 9 ). - Plot the relative fl uorescence units (FU) versus the total
amount of AMC (μmoles per well; [AMC]/5,000 to give total
moles AMC in 200 μL well). - Fit a linear regression line to this data with the formula
y = mx + b , where y is FU and x is μmoles of AMC per
reaction. - Using the AMC standard curve, calculate the amount of AMC
released (increase in AMC; Δ AMC), using the Δ FU value
obtained earlier from the plot of FU versus time. Ensure the
amount of AMC is in μmoles. - Divide Δ AMC by T (in min) to give the number of enzyme
units ( U ) in your 200 μL well ( U = amount of substrate released
in μmol/min). - Divide the number of enzyme units ( U ) by the amount of pro-
tein (in mg) in the reaction to give the specifi c activity ( U /mg).
3.5.2 AMC
Standard Curve
3.5.3 Converting Relative
Activity to Specifi c Activity
( See Note 19 )
Trypanosoma Metacaspases