How to determine low Km values? - (Oct/01/2014 )

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
 

A couple of possibilities:

You could try one of the integrated Michaelis-Menten methods which regress the amount of substrate per time of an individual assay as opposed to the rate per concentration of traditional nonlinear regression.

Getting a quicker/accurate ‘start’ on your reaction should help. The ultimate solution for this is stopped-flow equipment but often refining you technique for starting the reaction in a cuvette will be sufficient. The best I have is an altered inoculation loop which will hold 50uL and will add starting reagent and mix the solutions in a single dip.