Zlotnikov, I.D.; Ezhov, A.A.; Kudryashova, E.V. Intermolecular FRET Pairs as An Approach to Visualize Specific Enzyme Activity in Model Biomembranes and Living Cells. Biophysica2024, 4, 340-356.
Zlotnikov, I.D.; Ezhov, A.A.; Kudryashova, E.V. Intermolecular FRET Pairs as An Approach to Visualize Specific Enzyme Activity in Model Biomembranes and Living Cells. Biophysica 2024, 4, 340-356.
Zlotnikov, I.D.; Ezhov, A.A.; Kudryashova, E.V. Intermolecular FRET Pairs as An Approach to Visualize Specific Enzyme Activity in Model Biomembranes and Living Cells. Biophysica2024, 4, 340-356.
Zlotnikov, I.D.; Ezhov, A.A.; Kudryashova, E.V. Intermolecular FRET Pairs as An Approach to Visualize Specific Enzyme Activity in Model Biomembranes and Living Cells. Biophysica 2024, 4, 340-356.
Abstract
There are several well-known methods for determining enzyme catalytic activity, including spectral techniques and electrochemical approaches. However, at present, there is no definitive solution to the challenges associated with the fluorescent approach in, for instance, systems of reverse micelles, non-aqueous media, and model biomembranes. We propose the use of the fluorescent resonant energy transfer (FRET) technique, which can enhance the selectivity of fluorescent detection of enzyme activity by shifting the maximum emission wavelength to the longer-wavelength range and reducion the influence of medium components. As fluorescence donors, we have used derivatives of 4-methylumbelliferone (MUmb) and 7-amino-4-methylcoumarin (AMC), which also serve as substrates for hydrolases such as chymotrypsin, phosphatase, and asparaginase. During the catalytic depletion of substrates, an accumulation of a highly fluorescent product is observed. This product can be specifically detected using the fluorescent marker rhodamine 6G (R6G), which emits light in the red region of the spectrum (550–600 nm) with high quantum yield. A significant FRET effect is observed when forming AOT reverse micelles. This is due to the proximity of fluorophores within the hydrophobic core of the micelle, which is shorter than in a buffer solution. This approach have practical implications for visualizing intracellular processes. The FRET technique can be used to determine intracellular enzymatic activity by detection with confocal laser scanning microcopy (CLSM), as demonstrated with L-asparaginase, a clinically important anti-leukemia medication. The approach suggested will allow researchers to study enzyme activity within cells. This has the potential to revolutionize medicine by improving drug development and shedding light on previously unknown aspects of cellular metabolism.
Biology and Life Sciences, Biology and Biotechnology
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