Protocol Online logo
Top : New Forum Archives (2009-): : Biochemistry

Km calculation - (Mar/31/2014 )

Hello everyone!!

I'm trying to measure the km of MMP2 in the presence of a peptide substrate. I measured the increase of fluorescence at various concentrations of substrate but I encontered some problems with high substrate concentrations. High substrate concentrations cause the saturation of the instrument signal and the increase of fluorescence is no more visible. Lowering the sensibility of the instrument signal could help to solve the problem for high concentrations, but it could be  problem for the lower ones.

Do you know how to skip this problem?


Thanks for any help!!



if all you want to determine is km then you need to determine vmax. you can sacrifice the lowest concentrations because you shouldn't need them for km determination.


Look at using one of the ‘integrated-MM’ techniques. I have an Excel version of the Schnell & Mendoza equation but there are others dating back to the 70’s. They follow the progress of the entire curve at any given substrate concentration to estimate the Km and Vmax.  I am still not convinced they give as good an answer as the traditional method of varying substrate concentration but in your situation you may not have a choice.