spectrophotometry enzyme substrate dilution problem

[stinkytofu]

we wanted to see if different [substrate] affected velocity of rxn with an enzyme

We kept the enzyme volume always 0.2 mL optimal pH is 8. We did everything at pH 7 and total volume in cuvet was 3mL

0.2 mL enzyme + 0.3 mL substrate + 2.5 mL tris = total volume always 3ml
0.2 mL enzyme + 0.9 mL substrated + 1.9 mL tris = total volume always 3ml
Etc..

We would put these cuvets under a spectrophotometer determined %T and graphed it.

Another group did the exact same thing so that we could compare our results to make sure they were similar.

But the group made an error. Instead of a total volume of 3mL they used a total volume of 5 mL

i.e.
0.2 mL enzyme + 0.3 mL substrate + 4.5 mL tris = total volume always 5ml
0.2 mL enzyme + 0.9 mL substrated + 3.9 mL tris = total volume always 5ml
Etc..

They added more Tris. Now their %T values (at 5mL total) are higher than our %T values (at 3ml) so they have a lower [ ] when I used beers law to determine the [ ]. this lower [ ] is really skewing my data.

information i have:
Enzyme = 0.135g/700 mL optimal pH is 8
Tris buffer = 0.05M
Substrate = 1mM

Can I do anything to fix this?
They numbers are really skewing my data. I would prefer not to exclude their data. Any suggestions?



thanks

[NATHANALANPATRICKROGERS]

Use excel to find the equation of the [two] sets of data and solve.

[stinkytofu]

maybe i'm an idiot but what would i be solving for?


when 0.3 mL substrate was added I graphed the formation of [product ] vs time and calculated the slope which equals initial velocity.

Then for 0.9 mL substrate added I graphed the formation of [product] vs time to get the slope = intial velocity.

etc for each volume of substrate added.

then I graphed initial velocity versus [substrate] to determine the slope.

I did the same thing when the total [ ] was 5mL.



the intial velocity versus [substrate] for volume total 3 mL = 0.0112

the intial velocity versus [substrate] for volume total 5 ml = 0.0054

I don't know how to fix the difference in the numbers.

I was wondering if you could explain what I would be solving for?

I have everything in excel but still not too sure what to do with it.

thanks

[NATHANALANPATRICKROGERS]

There are a number of ways to do this but none are particularly eloquent - unless you have heaps of data and the answer is in fact plausible.

My suspicion is that the substrate -vs- reaction time graph for your enzyme may in fact be a squiggly line [Excel can produce a pretty good polynomial equation to reflect this though].