How to determine very low Km values?

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Daedalus348
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How to determine very low Km values?

Hello everybody,

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?

Best regards

EDIT: Sorry, just discovered that the substrate concentraion unit was missing. It's mikroMolar.