Bioassays Based on the Ultrafast Response of a Reporter Molecule
In this project we explore the capability of using ultrafast detection technologies for a fast analysis of biomolecular transformations. As an example the ultrafast response of TMR -labeled BSA as a function of different extents in proteolytic cleavage was investigated. We compared four samples of masses differing over several orders of magnitude: untreated, TMR-labelled BSA (66 kDa), TMR-labelled BSA treated with elastase (6-33 kDa) and with subtilisin (< 3 kDa), as well as the pure label TMR (0.4 kDa). A direct comparison with gel-electrophoresis revealed that various ultrafast parameters give robust information about the progress of the proteolytic cleavage. In the present example we found the ratio of the transient absorption signal observed at 0 ps and 50 ps after excitation (lPump = 540 nm lProbe = 570 nm) to be the most precise parameter determining the proteolytic cleavage. This parameter allowed to determine the mass accurately within 1 s (Z´-factor of 0.83) or within 600 ms measuring time per sample (Z´-factor of 0.64). This indicates that many of the known ultrafast detection technologies might be used for monitoring biochemical transformations probably even without any labeling procedure
Schematical presentation of processes contributing to the ultrafast response of a fluorescence label attached to proteins. Yellow arrows indicate VET, red arrows indicate protein and solvent reorientation. Shorter arrows refer to slower processes.
For details see:
C. C. Quentmeier, A. Wehling and P. J. Walla, “A bioassay based on the ultrafast response of a reporter molecule”, Journal of Biomolecular Screening 12 No.3, 341-350, (2007).