I have used French press to disrupt bacterial cells. After ultracentrifugation, the supernatant was used for determination of enzyme activity (PQQ-Glucose dehydrogenase).
Now, I would like to determine the effect of some inhibitory compounds on this enzyme. Should I also purify this enzyme or is the crude extract enough?
Dear Rasoul Shafiei. As i know, Its always reccommended and a common practise to study /determined kinetic paramters with the enzyme of highest purity. However, at preliminary level,enzyme activity can be checked in the crude extract and compare the enzyme activity of the purified enzyme to get some idea about specific activity folds. Well as you mentioned that you determined the enzyme activity with the crude extract. You may also check inhibitory activity of with the crude extract,however it may not give you the accurate measures or the inhibitory effect of any compound,since other impurities may hamper the inhibition potency of any chemical. Therefore my suggestion would be patient, get your protein/enzyme purified using suitable method (Ni-His based affinity Chromat,Gel Filteration, MonoQ/S Ion exchange Chromatography) and then study its inhibition kinetics. You may get more precise and accurate kinetic paramters (Km,IC50 etc). Correct me, if i am wrong.
Good Luck in your research endevours! Cheers
Regards,
Irshad Baig
PhD Student
Hello, thanks.
In fact I want to compare the PQQ-GDH activity of a bacterial during its growth phase (Log, early and late stationary). Then the inhibitory effect sof some produced metabolites should be tested on those samples.
So, can I use the crude extract to compare the differences between samples?
If you know that the organism produces only one enzyme with the activity you want to measure and if you have an assay that is highly specific for that enzyme, you might use the crude extract to get an initial indication of the effectiveness of the inhibitor, or, as you wrote more recently, to compare the activity in different stages of cell growth. However, as Irshad wrote, in all cases the preliminary experiments should be follow up with studies using the purified enzyme.
If you have a good and specific assay for your enzyme, then you can use a crude enzyme preparation to COMPARE the inhibitory effect of compounds. You can even study inhibition kinetics. With purified protein you can do binding studies without fear of competing proteins, but real life involves cells (ie crude preparations!).
The first question you have to answer is how reliably you can measure the activity of your enzyme in trhe crude extract, regardless of inhibitors. If you can get good numbers without undue interference from other activities in the extract, then you need to think whether there is any reason why other components in the crude extrtact might either destroy your inhibitor or just bind and sequester it making it unavailable to your enzyme. Assuming the answers to those questions are provisionally encouraging, give it a try. If there are already known inhibitors, it would be a good idea first to see if you can demonstrate that predicted inhibition using the crude extract. If not, then just try. It could give you a convenient rough screen to select those compounds that are worth looking at further. However, for good quantitative data for those compounds you eventually decide to concentrate on, I would always want the final experiments to be done with the pure target enzyme
No, It would always be better to use the purified enzyme to check inhibitory activity of the compounds. Even if you have determined the enzyme activity in the crude extract, inhibitory activity has to be checked with the purified enzyme preparation due to the following reasons.
1. Possibility of the binding of inhibitor in question to other proteins present in the crude extract, which may limit the inhibitor concentration available for the enzyme. Therefore, you may not see any effect of the inhibitor. However, the inhibitor is not available for the enzyme.
2. Since these inhibitors are metabolites as you say, possibility is there that they might be acted upon by other enzymes available in the extract.
Therefore, in order to confirm the inhibitory action of a molecule, use the purified enzyme and compare it with the control.
You can purify the enzyme from the extract by affinity purification in a single step, if possible.
Hello, Thanks.
So, I should purify the enzyme to get precise results.
Could you please give me some information about PQQ-GDH?
Which kind of column is suitable for purification?
Dear Rasoul, Please see the following reference for purification.
Biotechnology Letters 22: 1343–1347, 2000.
Paul Engel's answer cannot be bettered. He very clearly points out the possible uncertainties with working on the impure enzyme and extract but it is certainly worthwhile doing some preliminary studies with the crude material so that when you have the pure preparation you will already have some idea of the likely potency of the inhibitory material This will be useful for designing rigorous studies of the inhibition.
Peter Butterworth
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