Two‐Color Two‐Photon Excitation of Intrinsic Protein Fluorescence: Label‐Free Observation of Proteolytic Digestion of Bovine Serum Albumin

Abstract

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.

Monitoring of Demasking of Peptide Bonds During Proteolysis by Analysis of the Apparent Spectral Shift of Intrinsic Protein Fluorescence

“…As measuring parameters, intensity and fluorescence decay time have been previously used. 5,6 The measurement of the emission maximum and its shift with proteolysis presented here is new. The measurement of fluorescence intensity is considered less appropriate because it is influenced by many different factors, while both the decay time and the shift of the maximum appear to be more specific.…”

“…4 Fluorescence spectroscopy was also used for the monitoring of proteolysis processes by the measurements of the changes in intrinsic tryptophan fluorescence. 5,6 These studies engaged different spectroscopic methods, including simple measurements at one wavelength of the changes in static fluorescence intensity of Trp residues 5 , or much more complicate technique based on the determination of the apparent life time changes with two-color two-photon laser equipment. 6 In many cases, extrinsic fluorophores with emission in the visible range of the spectrum are used which can be easily measured.…”

“…5,6 These studies engaged different spectroscopic methods, including simple measurements at one wavelength of the changes in static fluorescence intensity of Trp residues 5 , or much more complicate technique based on the determination of the apparent life time changes with two-color two-photon laser equipment. 6 In many cases, extrinsic fluorophores with emission in the visible range of the spectrum are used which can be easily measured. Measurements of the fluorescence of extrinsic fluorophores attached to the protein substrate was also used for monitoring of proteolysis.…”

Applications of the time-resolved two-colour two-photon excitation of UV fluorophores using femtosecond laser pulses

“…As mentioned above, one of the first applications of 2C2P fluorescence was the label-free monitoring of a proteolytic digestion of the protein BSA by subtilisin [15]. BSA contains five tryptophans, which act as a natural built-in probe for their surroundings.…”

“…In addition to the above-mentioned proteolytic cleavage of BSA by subtilisin, a similar reaction between BSA and elastase is monitored. The experimental setup is shown in figure 1 and was described in detail previously [15]. The reaction is performed at concentrations of 0.1 mM BSA and 0.01 mM elastase.…”