Berlin 2001 – wissenschaftliches Programm
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Q: Quantenoptik
Q 32: Poster: Laser Spectroscopy
Q 32.9: Poster
Freitag, 6. April 2001, 12:30–15:00, AT2
Detection of sub-populations of protein-bound sensor molecules via ESA spectroscopy — •Gisela Grunwaldt1, Sophie Haebel2, Christian Spitz1, Martin Steup3, and Ralf Menzel1 — 1Institute of Physics, Photonics, University of Potsdam — 2Center for Biopolymers, University of Potsdam — 3Institute of Biochemistry and Biology, Plant Physiology, University of Potsdam
Extrinsic covalently bound labels that react sensitively to their microenvironment have become important tools in studying proteins. Conjugated to proteins of interest they monitor conformational changes, interactions with other molecules and related processes. To our knowledge excited state absorption (ESA) spectroscopy has not yet been exploited to obtain data on the microenvironment of such protein labels.
We characterized a variety of fluorescein isothiocyanate-myoglobin conjugates with respect to degree of labeling and localization of the labels on the protein. We furthermore measured their quantum yield and lifetime of fluorescence and recorded their ESA spectra under different experimental conditions.
The conjugates were shown by mass spectrometry to be highly heterogeneous regarding the degree of labeling and localization of the label. ESA spectra of untreated conjugates revealed two clearly separated absorption maxima. The partly denatured conjugate as well as the free fluorophore showed only one ESA maximum. We conclude that there are in untreated conjugates at least two sub- populations of bound fluorophore distinguishable by ESA. ESA measurements, therefore, provide useful additional information on the microenvironment of protein-bound fluorophores and can become a powerful tool for future investigations on protein properties.