Enzyme Reaction Rate

[Dr. R.J. Beynon]

Re: Lineweaver-Burk plot etc for determination of Km, Vmax

I suggest you have a look at a book - Colquohon, D. 
"Lectures on Biostatistics" (1971) Clarendon press, Oxford

this shows beautifully how, of all the graphical methods,
the L-B gives the worst, and most biased values of Km and Vmax.

Two better solutions are:

The "Eisenthal" plot for parameter free (median-based estimates)
-can be graphical, but better done by computer.

Non-linear curve fitting, using gradient (Marquardt, usually)
or direct (Simplex, patternsearch) algorithms. I have found that
with well-behaved data, they give identical results. There is a good
deal of software around, but my choice would be P.Fit by Biosoft.
It gives superb analyses/stats and the user interface, although
idiosyncratic, is fast and efficient. There are others - what platform
will you use?

Incidetally, Excel (latest version) has a good solver that is a gradient
optimiser. I have used this to find Vmax and Km from kinetic data/

I once wrote a chapter for a book in which I described the background to
curve fitting. I could let you have this if you were nterested.

Rob Beynon
Proteolysis research Group, Biochemistry  Liverpool,UK