I have a sample rich in proteases. I would like to know if they are metaloproteinases or serineproteases.
Many papers employ EDTA or o-phenantroline for metaloproteinases inhibition and PMSF for serineproteases, by zymography.
However, some papers pre-incubate the enzyme with these inhibitors and then run the gel (option 1). Other authors, put the inhibitors in the reaction buffer (after renaturation with SDS) (option 2)
My question is: which is the best choice, option 1 or 2?
Regarding option 1 (pre-incubation), in view of the mechanism of inhibition of these inhibitors, there is a risk of the inhibitor do not act? Or even, in the renaturation step, may the inhibitor be lost?
Please, help me with this doubt.
Thanks in advance,
Juliana.
I have not tried this myself, but your doubt seems to be justified - the inhibitors you mention are not covalent, except for PMSF, and so could easily drop off in detergent, and so it makes more sense to make sure the inhibitor is added and present when you actually want to detect the activity or the inhibition of activity. However, I think there is one other thing to worry about and that is the possibility of proteases digesting themselves or one another in the process of preparing them for SDS gels electrophoresis. A half-unfolded protease presents a very nice substrate for a still-folded protease! This means you need to be careful with the timing and method of sample preparation and play with conditions until you find what gives reliable reproducible results.
In the case of PMSF, because it is covalent, there can be no serine protease activity after renaturation if you have used PMSF beforehand, so surely you want to compare the results on the zymogram plus/minus PMSF added only AFTER renaturing ?
Hi Juliana, I did the two options, but I worked with aprotinin instead of PMSF. Best regards
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