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Lipase surface diffusion studied by fluorescence recovery after photobleaching
Andreas W Sonesson1, Thomas H Callisen, Hjalmar Brismar
1YKI, Institute for Surface Chemistry, Stockholm, Sweden.
Langmuir : the ACS Journal of Surfaces and Colloids
|December 1, 2005
Summary
We developed a novel method to study enzyme diffusion on surfaces. Thermomyces lanuginosa lipase (TLL) diffusion was slower on hydrophobic surfaces, influenced by its structural changes upon adsorption.
Area of Science:
- Biochemistry
- Surface Science
- Enzyme Kinetics
Background:
- Understanding enzyme behavior on surfaces is crucial for biocatalysis and biosensor development.
- Surface properties significantly influence protein adsorption and diffusion dynamics.
Purpose of the Study:
- To analyze the surface diffusion properties of Thermomyces lanuginosa lipase (TLL) on different silica surfaces.
- To develop and validate a novel method for measuring diffusion on solid surfaces using fluorescence recovery after photobleaching.
Main Methods:
- Developed a novel 1D fluorescence recovery after photobleaching (FRAP) method using confocal microscopy.
- Calibrated fluorescence intensity to lipase surface density using ellipsometry.
- Measured diffusion coefficients and mobile fractions of TLL on hydrophilic silica, DDS-silica, and OTS-silica surfaces.
Main Results:
- The novel FRAP method was validated using bovine serum albumin diffusion on glass.
- TLL exhibited the lowest diffusion coefficient and mobile fraction on the hydrophobic octadecyltrichlorosilane (OTS) surface.
- Diffusion rates decreased over time on hydrophobic surfaces, correlating with TLL's conformational changes.
Conclusions:
- Surface hydrophobicity significantly impacts TLL diffusion dynamics.
- Adsorbed TLL's conformational transitions (closed to open, hydrophobic state) are critical for its surface diffusion behavior.
- The developed FRAP method offers a robust approach for analyzing surface diffusion of biomolecules.