Hello
I would like to measure the ACE inhibitory activity of hydrolysate using FAPGG substrate according to this method. Do you think this is a good method?
In brief, FAPGG (0.5 mM) and various
concentrations of the samples were completely dissolved
in 50 mM Tris-HCl buffer (pH 7.5). 20 μL of ACE (1 U/mL
dissolved in 50 mM Tris-HCl buffer) were then mixed with
200 μL of samples of various concentrations as experimental
samples, or mixed with 50 mM Tris-HCl buffer as a negative
control. Following the addition of 1 mL of 0.5 mM FAPGG to
the reaction mixture, the optical density was determined after
0 and 30 min at a wavelength of 345 nm. The ACE inhibitory
activities were expressed as the 50% inhibition concentration
(IC50). The values of percentage inhibition were then calculated
using the equation:
Inhibitory activity (%) = {1 – (ODsample/0min – ODsample/30min ) /
(ODcontrol/0min – ODcontrol/30min)} × 100.
Like Chibuike C Udenigwe said you should consider incubating the reaction mix at 37ºC but you should also be aware of the absorbance of your inhibitor since if it overlaps with your substrate it will create interference.
Also you might not need to run your reaction for 30 mins. If you can measure the absorbance every minute over 30 minutes to find how long it takes for the reaction to reach a steady state and that's approximately how long you need to measure. This will also help you to find a linear portion of the kinetics curve to calculate the initial velocity of the reaction.
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