Hello,
I am trying to calculate the kinetic parameters for my enzyme using fluorescence of the substrate and product.
The AUC for the product is not showing much difference, when I freeze and store the reactions. What could be the reason?
Also, the AUC of the substrate shows a gradual change with time. But, when I plot the AUC against time, I am getting a negative slope for the rates.
How can I plot a michaelis-menten curve with this? How is that negative slope data analyzed for calculating the kinetic parameters (representative image attached for the graph of FL vs Time)?
Thanks
Whether the signal used to follow the reaction increases or decreases with time depends on the specific signals of your substrate and product - for example, if the specific fluorescence of your substrate is higher than that of your product, you expect the signal to decrease as the reaction progresses, the substrate concentration decreases and the product concentration increases.
The specific fluorescence of the reactants can be used to determine actual concentrations at the different timepoints, and from the slope of the curved determine the actual reaction rates.
You plot these reaction rates as a function of substrate concentration to get a Michaelis-Menten plot, which allows you to visualise the Km as the substrate concentration at which the reaction rate is half the maximal rate, while at very high substrate concentrations the curve asymptotically approaches the rate Vmax.
You can either computationally fit the Michaelis-Menten equation, or, for graphical determination of the values plot the data as a Lineweaver-Burk plot.
https://www.youtube.com/watch?v=4aVOtcpGaZc
Some chromogens are detected after the substrate conversion so you monitor the product production, which turn into positive slopes (more chromogen over time). In your case is the opposite, your chromogen is detected before the conversion into product so you are monitoring the substrate presence, turning into negative slopes (less substrates over time). You only have to take the absolute value of your slopes and calculate the enzymatic speed required to determine Km and Vmax using Michaelis-Menten equation.
- Badges
- Science topic
- Similar topics
- Engineering
- Modeling