No enzyme Activity (dioxygenase assay) - (Apr/01/2010 )

Hi guys,

I am assaying for a dioxygenase activity on a aromatic compound , basically a aromatic ring hydroxylating activity.

The cell was grown using the aromatic compound as sole carbon source .

during the mid log phase, i harvested the cell . centrifuged at 4000rpm at 4C for 30 min.

washed with 20mM K/Na Phostphate buffer once

then eventually the cell was resuspended in 50mM Tris-HCL pH 7.5 with 1mM DTT and 10% Glycerol

in order to extract the protein , i used a Vibracell 600W. 10% amplitude 2.5pulse and 2.5 pause. for 10 min. done in ice bath

The lysate is clear. then i proceed to centrifuge at 12,000rpm for 30 min.

The supernatant would be the cell-free extract in my stage.


So from here the reaction goes like this


1ml reaction (containing)

0.4mM NADH
0.2mM Ferrous ammonium sulfate
25mM Tris-HCL pH7.5
0.05 ml Cell Free extract
1mM of aromatic compound


incubate at 30C for 30 min

negative control is the similar reaction but without the substrate.


i compared the abs at 340nm for reduction in NADH.

at the end , i couldn't observe any significant difference.


What could be the problem.

any suggestion would be highly appreciated

1. extracellular enzyme??
2. denatured protein?
3. any other??

it's my first time experience in doing enzyme assay . hope to hear from you guys soon.

did you run a gel of your preparation? did it show a band or pattern that you expect for your protein?

you could have a denatured enzyme. you may also be experiencing the presence of a native inhibitor. the enzyme may be too dilute in the extract.

you may have to at least partially purify the enzyme.

mdfenko on Apr 1 2010, 11:24 AM said:

According to the literature this sort of dioxygenase system is a multicomponent one. IN any case, from what i have read most group could observe activity when they did the enzyme assay with cell-free extract but using other sort of aromatic compound.

As of now, i do not know whether which enzyme is specific for its reaction. The fact that it could grow on the compound as its sole nitrogen /carbon source should indicate it has the pathway for this degradation. so in my mind, the enzyme(s) should be present.

Currently hoping to develop/modify an existing assay first to determine its activity prior to partial purification.

Thanks for the comment.

i have added glycerol and dtt. i wonder if you have any suggestion as to how to stabilize and prevent its denaturation.

In addition, how to prevent the effect of native inhibitor? that's somewhat new to me

hanming86 on Apr 1 2010, 11:51 PM said:

it depends on what is causing the protein to denature. sometimes using additives will protect. sometimes adjusting extraction conditions will help. sometimes a combination will help (sorry if this appears to be evasive, but, i just don't know enough about what you are doing to be specific).

you can eliminate native inhibitor(s) by partially purifying. you may only need to perform an ammonium sulfate fractionation to eliminate all or enough of the inhibitor to reveal the activity. or fractionation on a gel filtration column may help.