I'm trying to assay different lipases and their kinetic parameters using Ellman's reagent and DMPTB as a substrate.
I've tried to measure the rate of reaction using a microwell plate in a spectrophotometer.
My protocol consists of making a premix with 0.5M Tris, 0.1M EDTA, 5% Triton, MQ water and 40mM DTNB. I'll add specific volumes of the DMPTB, mix it with the lipase and then assay it using the microwell plate. I have found that there is not colour change in the assay unless the DMPTB concentration above 5mM, so I have tried assaying different concentrations of DMPTB between 5-20mM.
However, at increasing concentrations of DMPTB (above 10mM) the rate of reaction does not increase with DMPTB (i.e. not following M-M laws)
I have also noticed precipitation in DMPTB stock solution (dissolved in Triton and 50mM Tris). But I vortex the solution prior to pipetting to offset this effect. Could this be contributing to the problem I'm seeing?
Any help would be great !
Hi there,
The M-M equation states that at saturating concentration of substrate the rate of reaction reaches a limit.
What is the composition of the assay? Your premix seems highly concentrated in Tris EDTA and Triton.
What source of lipase do you use and how active is it?
I agree with Dominique Liger , your substrate conc seems very high and might be expected to saturate the enzyme. It is hard to see why you would need so much substrate ; DTNB product has a molar extinction coefficient of about 13800, which means in a typical plate assay you’ll get an OD of around 1 with 100uM thiol. Thus substrate concs in the range 250uM to 10mM should be perfectly fine to generate sufficient product for detection in the O-2 OD range, while still representing only a SMALL % conversion to product (essential for a valid kinetic study). I get the impression your enzyme activity is probably low. It is also worth checking that your pH is appropriate for a DTNB assay. Needs to be 7.5 or higher.
Dominique Liger Nick Gee Thank you both! I replaced the premix with one at pH 8 and removed the EDTA, and now can see colour change within in the 250uM - 5mM range.
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