So, I'm doing an enzyme assay and there are two compounds in the assay, one a product and the other a cofactor, that absorb at the same wavelength. It makes it very difficult for me to detect activity because I am measuring depletion of the cofactor to get kinetic data, but its signal is overshadowed by absorbance from my product. Do you have any suggestions as to how I can address this problem? I need to subtract the absorbance of the product from the absorbance of the cofactor. I have the molar absorptivity of both. Thanks!
Hi there,
So OD results from the sum of both contributions. If product and cofactor absorb differently then you may calculate a proper reaction velocity. A t=0, OD=epsilon.l.[cofactor]0 and at a given time OD=epsilon.l.([cofactor]0-[product]) +epsilon'.l.[product] so OD=(epsilon'-epsilon).l.[product] + epsilon.l.[cofactor]0=delta epsilon.l.[product] + ODt=0. So if you know both epsilons and that they are significantly different you have a linear relationship between OD and [product]. Here I considered a 1:1 stoechiometry between cofactor and product. if it's not the case, the term [cofactor]0-[product], which represents the concentration of cofactor at the given time, has to be corrected accordingly. For instance if 2 cofactors are needed to get one product then the term becomes [cofactor]0-2[product].
Take absorbance spectrum of the cofactor pror to the reaction and after the reaction, the substrate, and the product separately and overlap them. The goal is to identify a wavelength at which the change in absorbance of the cofactor before and after reaction is maximal and the change in absorbance of the substrate before and after reaction is minimal. This is the wavelength to monitor. Then prepare a standard curve in which the proportion of post-reaction cofactor to total cofactor increases and the proportion of product to product+substrate increases stoichiometrically. Use the standard curve to figure out how far the reaction has proceeded.
Alternatively, try to find a wavelength at which the absorbance increase of the product relative to the substrate is maximal and the absorbance decrease of the cofactor is minimal. Monitor that wavelength instead. It is actually preferable to monitor the increase is product formation rather than the decrease in cofactor concentration because the signal-to-background ratio is higher.
Finally, it may be best to monitor two wavelengths simultaneously if the instrument has that capability. The ratio of absorbances at the two wavelengths can be used to increase the signal-to-noise ratio and may alleviate the interference between the two substances.
Hi
I guess u r using a control (blank) which is without a product so add cofactor plus enzyme to the control and measure the OD along with assay tubes in this case any increase or decrease in OD is for the product.
The other thing that you may try is the GC (gas chromatography) to measure the formed product.
Hope this helps
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