enzyme kinetics help!!

[ellie9922]

Okay, so I'm trying to work out the effect of substrate concentration on reaction velocity.
I did an assay, using PNPG in varying concentrations. I then need to work out the reaction rates in order to graphically determine the Km and Vmax for each reaction.

So, my results are:
(volume of PNPG & absorbance reading)
0.1 (PNPG) = 0.078 (absorbance)
0.2 = 0.124
0.3 = 0.268
0.4 = 0.315
0.6 = 0.507
0.8 = 0.632
1.2 = 0.724

To convert into concentration, I used the Beer-Lambert law. I had already calculated the conversion factor, of 333.3, from a previous experiment.

This gave me concentrations of:
25.997
79.992
89.324
104.990
168.983
210.646
241.309

I need to know how to convert this into initial reaction velocity in nanomoles per minute.
I believe, as I used 10ml and incubated the solutions for 5 minutes each, I have to times by 10 and divide by 5.
So, for the first tube: 26 x 10 = 260 and /5 = 52

I would really appreciate it if somebody told me if i was right or not. As when I attempt to plot graphs using this data, for example an eadie-hoftsee plot, it just isn't looking right.

As I have to plot [ S]/v against [ S].
So, every x value on the graph would therefore be 2. Which cannot be right?

as [ S] = 26
and v/[ S] = 52/26 = 2