[JCK]

Hi
I'm currently researching the enzyme kinetics of alkaline phosphatase on p-nitrophenyl orthophosphate.  The product formed p-nitrophenol (mol/L/min) appear to be low.  I'm wondering if daylight or other factors could have effected the enzyme and as a result the low rates can be accounted for.  We had added glycine buffer to maintain pH.  A salt soluntion containing magnesium and zinc ions as cofactors was also added.  The practical was carried out at room temperature.  100 microlitres had been added to all assay solutions but the volume used of PNP was increased in all 6 cuvettes.  Some time had passed from when the technicians had set up the practical and the lecturer had finished explaining what we had to do and I have been wondering how this could have effected the enzyme?  If anyone can point me in the right direction that would be great.  Thank you.

[Arkcon]

I'm currently researching the enzyme kinetics of alkaline phosphatase on p-nitrophenyl orthophosphate.

A little old fashioned, but ya gotta start somewhere.  Sounds like fun.

The product formed p-nitrophenol (mol/L/min) appear to be low.

 

Bummer.

I'm wondering if daylight

umm...no

or other factors could have effected the enzyme and as a result the low rates can be accounted for.

you'll have to find something else, yes.

We had added glycine buffer to maintain pH.  A salt soluntion containing magnesium and zinc ions as cofactors was also added.  The practical was carried out at room temperature.  100 microlitres had been added to all assay solutions but the volume used of PNP was increased in all 6 cuvettes.

If that's what the protocol said, you should be fine.  You might want to double check.

Some time had passed from when the technicians had set up the practical and the lecturer had finished explaining what we had to do and I have been wondering how this could have effected the enzyme?  If anyone can point me in the right direction that would be great.  Thank you.

You can try and work this angle as you see fit.

Not much to talk about here really, it's a simple assay.  People get a little sloppy with enzymes sometimes, treating them like chemicals.  They are, but as they've come from living things, you have to treat them as such -- not exposing them to bad storage conditions, adding supporting reagents properly, careful measurements with reagent additions, that sort of thing.  Try and see what your notes can tell you.