Hello everybody

I am now working on one membrane bound enzyme for which L-malate works as a substrate. To calculate the Vmax and Km value for the substrate, I have done an experiment in Plate reader with different concentrations (8 concentrations including the blank and tetra-plicate) and recorded the absorbance for each concentration. In each well I used a total of 200ul of solution containing 5ul of enzyme (fixed for all well), 190ul of reaction mixture (including DCIP) and 5ul of the L-malate and run the experiment at 600nm for 23 minutes (3 minutes for background without the substrate and 20 minutes for activity with L-malate). However, after getting the absorbance for all concentrations, now I am in confusion, in fact helpless, how to calculate the initial velocity for each concentration, as probably (so far I read) I am not able to use the Beer-Lambert law because I dont know the "Extinction co-efficient" for DCIP in case of Plate reader (I know in case of 1 cm cuvette). I think as the path length for the Plate reader is not known and the direction of the wavelength is different from spectrophotometer, so I dont know how to calculate the initial velocity.

One of my lab mate suggested to use the Lineweaver Burk plot, using the delta absorbance at certain time (the reading of the absorbance at certain time, in the present study at 3.5 mins) as V and prepare the plot. I did the same and calculated Vmax and Km, but my supervisor informed that the absorbance cannot be used as velocity. So, I am wondering how can I calculate the velocity from this absorbance. In this context, I am asking a detailed explanation how can I calculate the initial velocity and Vmax and Km.