Has anyone measure the neutrophil elastase activity in plasma by using the N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide substrate? I tried following differents papers instructions, but they were pretty incomplete:
I tried with 50µl of 2mM substrate + 50µl of plasma during 15 minutes (37ºC) and then reading 405 nm, the same during 2 hours at 37ºC and the results made no sense (I didn't even notice a color change).
I also tried with 200µl of 2 mM substrate + 25µl of plasma (following a different paper), it only make sense if I correct the 405 nm with 490 nm absorbance, but I am not sure if that makes any sense.
Thank you!!
Hi Adam,
You're right, there is the data of one of the assays, thank you!
Try collecting time-based data, at 1 minute intervals, for example. It will be easier to see how much of the absorbance is due to catalysis (it increases with time) and how much is due to background (it is constant with time).
I am not sure if will be able to neutrophil elastase activity in plasma using chromigenic substrate as you mentioned. Hne activity in blood is strictly controlled by inhibitors. One solution is use larfe excess of substrate over enzume inhibitor complex since in this form majority of Hne is present in plasma. Other way is to use fluorgenic or fret substrate which is much more sensitive than one you are using . Good luck
If NE activity in plasma is inhibited, as mentioned by Adam Lesner, then measuring the catalytic activity will not be a good indication of the amount of the enzyme present. Instead, you could do a quantitative immunological measurement, such as an ELISA, using purified NE as a standard.
Thank you both so much, Adam B Shapiro and Adam Lesner !!
Sensi de los Reyes-Gª Pastor Have you got the results of neutrophil elastase activity in plasma? I'm doing the same work. Could you kindly to tell me how to measure the NE activity with so many incomplete papers? 3q
- Badges
- Science method