I am currently characterizing several purified enzymes and have encountered a problem.
I am determining Km and Vmax with nonlinear regression, but the Km values of my substrates are very, very low (range of 2-20 mikroMolar).
My problem is that it is very hard to measure these low substrate concentrations with a UV-spectrophotometer and thus following it is nearly impossible to get accurate data for Km and Vmax with nonlinear regression. At least I think it is.
I could try another analysis method like Lineweaver-Burk, but I think this method is a bit out-dated.
What can I do? Are there any other methods to determine very low Km and Vmax values?
EDIT: Sorry, just discovered that the substrate concentraion unit was missing. It's mikroMolar.