Question

I measured some enzyme kinetics in a practical course using a substrate-based FRET assay. Unfortunately some of my plots show weird effects. There was always a decrease in signal after 35 minutes. But the curve always regained. During the whole measurement the plate was placed in the reader, and was heated to 37°C.

x axis - time, y axis - fluorescence

My question is: Does someone know how/why this effect occurs?

Answer 1

An unexpected decrease in fluorescence signal after 35 minutes in your FRET assay could be caused by photobleaching, environmental or instrumental fluctuations, or temporary changes in the enzyme activity.

Step-by-step explanation:

You are observing a decrease in fluorescence signal after 35 minutes in your enzyme kinetics study using a substrate-based FRET assay, which then regains in your enzyme-catalyzed reaction. This effect could be due to a variety of factors. One possible explanation is the photobleaching of the fluorophores used in the assay, which can temporarily reduce fluorescence intensity.

As the plate is constantly heated to 37°C in the reader, this could exacerbate photobleaching or other environmental or instrumental fluctuations. Another possibility is enzyme instability at the given temperature over time, or the reversible formation of enzyme-product complexes, causing temporary changes in enzyme activity.

During the initial reaction rate (v0) determination, it is essential to consider these effects, as they can impact the accuracy of kinetic parameters like the Michaelis-Menten constant (Km) and the maximum reaction velocity (Vmax). Ensuring that your experimental conditions maintain enzyme stability and that the plate reader's performance is consistent over time is critical for reliable results.

Moreover, the enzyme concentration and substrate concentration should be optimized to mitigate such effects and improve the assay's sensitivity.