I am trying to express and purify F420-dependent enzyme. I monitor enzyme activity by doing a quick reaction of the enzyme with the cofactor and substrate (G6P). When I checked the activity without substrate or cofactor I still got activity. my lysis buffer contains 50mM potassium phosphate, DNAse I, glycerol, and BME. Even after trying to purify it using Ni-NTA column I got the same results.
I monitor the reaction at 420 nm because that's where I'm supposed to see a change
The reaction of this enzyme (EC 1.1.98.2), I believe is D-glucose-6-phosphate + F420(oxidized) 6-phospho-D-glucono-1,5-lactone + F420(reduced).
If you are monitoring the reaction at 420 nM, then you are following the reduction of F420. If you leave out the cofactor (F420), then there is nothing to monitor. Do you mean that you leave out Mg2+?
Have you ever done the experiment of leaving out the enzyme? Maybe the F420 is being reduced by BME. Have you ever tried leaving out the BME?
There is nothing in your mixture that absorbs at 420 nm (yellow color) except F420, so you should see just a constant baseline. Are you doing this measurement with a crude extract and crude fractions instead of a purified protein (hence the reason for the presence of DNase)? A crude extract will have some turbidity (cloudiness) which may clarify over time, causing the baseline to drift downwards. If that is what is happening, and if it is a large effect, it may not be practical to perform the assay on the crude extract. However, if you have a dual-beam spectrophotometer or if you are using a plate reader, you can simultaneously measure the absorbance of identical samples with and without G6P. The difference between the two samples would then be due to reduction of the F420, allowing you to follow the reaction of the enzyme.
Adam B Shapiro I’m trying to purify this enzyme. It’s a new project so there is no clear procedure. I expressed it in E. Coli and I get the cells, lyse them using the buffer mentioned and then load the cell lysate on Nickel column. Every step ( lysis, flow through from column, washing steps, and elution steps) is monitored using the activity assay as described. I form the baseline using with KPi buffer, G6P, and F420. the reaction is initiated by addition of a sample from each step. The problem is that I get a peak every time with and without cofactor/substrate
Alaa Aziz although it is an interesting question, you shouldn't bother with it too much, or you may never reach your results. Just use inactivated protein preparation as blank and compare them to native enzyme preparation.
When I was purifying zeatin isomerase, I've added the designated amount of my extract to buffer in two tubes. One tube I have boiled for 5 mins, the second I kept on ice. After cooling of both tubes, I added substrate and cofactor and measured activity in both. Then I substracted native - boiled and used that as proxy for activity of my enzyme.