[Goldie]

Hello,

I want to do a fluorometric P450 activity assay in which I should use glucose-6-phosphate dehydrogenase(G6P-Dh). My protocol says :
"The enzyme source was added to 50 µl of a reaction mixture (7-EFC 0.4 mm, glucose-6-phosphate 1 mm, NADP+ 0.2 mm and G6P-Dh 0.014 U in sodium phosphate buffer, pH 7.4)"
But the G6P-Dh data sheet says:
"To ensure maximum stability and recovery of activity, reconstitution with 5 mM sodium citrate, pH 7.4, is recommended. Phosphate buffer will inhibit the enzyme and should not be used"

Now, What should I do? Should I change the buffer in protocol to another buffer? If I do this, my enzyme source is a sample made in sodium phosphate buffer. Does it interfere with the reaction?

Thanks for your help

[Babcock_Hall]

There are at least two courses of action that you can take.  When there is a large dilution factor between the stock solution of enzyme and the assay solution, the concentration of the inhibiting substance falls by that factor.  Depending on its concentration and inhibition constant, this might or might not be enough to avoid inhibition.  The second thing that can be done is to do a buffer exchange step on a portion of the stock enzyme solution.  This can be done via a Penefsky column, via a commercial gel filtration column (such as a PD-10 column), or by dialysis.

[Goldie]

Thanks a lot!
The second method is not feasible for me but to increase dilution factor of the phosphate buffer, can I dissolve my G6P-Dh in Tris-Cl instead of phosphate buffer and add it to my sample (which already contains sodium phosphate buffer 0.1 M)?

[Babcock_Hall]

I can answer in general terms only.  When I have a new enzyme, I look in papers or other sources for what buffer it is stored in and what buffer it is assayed in.  Glucose 6-phosphate DH is often part of coupled enzyme assays, so there is probably an overwhelming amount of literature available.  Those points noted, I don't see a problem with Tris.  However, if the volume of enzyme solution is small, and the volume of assay buffer is large, then the assay buffer will be the major source of phosphate.  It seems to me that if the degree of inhibition from phosphate is not too great, then you might be better off tolerating it.  If phosphate inhibition is a severe problem, then you might have to change the assay buffer to Tris, HEPES, MOPS, BIS TRIS, or something else.