Two-Color Two-Photon Excitation of Intrinsic Protein Fluorescence: Label-Free Observation of Proteolytic Digestion of Bovine Serum Albumin
✍ Scribed by Stefan Quentmeier; Claudia C. Quentmeier; Peter J. Walla ; Karl-Heinz Gericke
- Publisher
- John Wiley and Sons
- Year
- 2009
- Tongue
- English
- Weight
- 317 KB
- Volume
- 10
- Category
- Article
- ISSN
- 1439-4235
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✦ Synopsis
Abstract
Two into one: Simultaneous absorption of two photons of different colors (2c2p) extends the applicable wavelength range of the Ti:Sa laser beyond that of conventional two‐photon excitation (TPE) into the UV region (see schematic). The intrinsic fluorescence of bovine serum albumin during proteolytic cleavage by subtilisin (see plot) are monitored without exposing the protein to damaging UV radiation.magnified image
Two‐color two‐photon (2c2p) excitation fluorescence is used to monitor the enzymatic cleavage of bovine serum albumin (BSA) by subtilisin. Fluorescence is generated by irradiation with spatially and temporally overlapping femtosecond laser beams resulting in simultaneous absorption of an 800 and a 400 nm photon. Thereby, excitation of the fluorescent amino acid tryptophan present in BSA corresponds to an effective one‐photon wavelength of 266 nm. The progress of protein cleavage is monitored by time‐resolved fluorescence analysis. The fluorescence lifetime of tryptophan decreases during the reaction. This demonstrates a novel label‐free multiphoton observation technique for conformational changes of proteins containing tryptophan. Due to the strong 2c2p fluorescence signal it is suitable for fast evaluation and monitoring of protein reactions. The course of the reaction is monitored simultaneously by gel electrophoresis. In contrast to conventional one‐photon techniques, 2c2p excitation enables label‐free protein fluorescence studies without irradiating the sample with UV light. Due to the dependence of the excitation on the power of both laser beams, excitation is limited to a relatively small focal volume. This results in dramatically reduced overall photodamage compared to direct UV irradiation. This method can be easily extended to microscopy imaging techniques.