A Simple Biophysical Experiment to Introduce Ratiometric Measurements and Microplate Fluorimetry in the Laboratory Class

Abstract

In the past decades fluorescence applications in the Life Sciences, Sensing and Nanotechnology had a huge burst, in part because a variety of instruments can be used to measure fluorescence, including fluorescence microscopes, flow cytometers, and high throughout microplate readers. Therefore, it is important to introduce students to different apparatus, underlying their common features and particularities.In many instruments, several experimental limitations hamper quantitative treatment of data, unless ratiometric measurements are made, i.e., the ratio of intensity at two different excitation and/or emission wavelengths, which eliminates a considerable number of artifacts. However, such methods are possible only when a spectral shift occurs.The denaturation of proteins often induces a blue-shift of the tryptophan residues emission, due to their increased exposure to the polar aqueous medium. Such shift permits the use of ratiometric measures to obtain the fraction of native and denatured protein, hence the denaturation curve.In this work, the denaturation of egg-hen-lisozyme by guanidine hydrochloride is studied. Since this process has been described by a two-state model, the analysis is straightforward. The students perform single-wavelength and ratiometric measurements, to observe the improvements brought by the latter. The experiment requires just the preparation of a series of solutions with protein and growing concentration of denaturant and respective blanks. With a 96-well plate it is possible to analyze 3-independent series of samples and test the reversibility of the process. From the denaturation curve, the students retrieve the free energy of lisozyme denaturation in water, and the cooperativity of the process. This work is performed successfully (Biochemistry Master Program), and the students reveal development of analytical skills and simultaneously deeper understanding of the physical-chemical principles behind protein structural changes.Acknowledgements: Ciencia2007, PEst-OE/QUI/UI0612/2011 and SFRH/BD/64442/2009 (FCT, Portugal).