Application of Ratiometric Measurements and Microplate Fluorimetry to Protein Denaturation: An Experiment for Analytical and Biochemistry Students

The number of applications of fluorescence spectroscopy in different areas chemistry has increased dramatically, in part because a variety of instruments are used to measure fluorescence, including high-throughput microplate readers. Therefore, it is important to introduce students to different instruments. With many instruments, several experimental limitations hamper quantitative treatment of data, unless ratiometric measurements, that is, the ratio of intensity at two different excitation or emission wavelengths, are made. However, such methods are not always applicable. The denaturation of proteins often induces a red-shift of the tryptophan residues emission. Such a shift permits the use of ratiometric measures to obtain the fraction of native and denatured protein. To our knowledge, the use of ratiometric analysis with fluorescence measurements obtained from a microplate reader for the study of protein (biomolecular) denaturation has not been applied as a teaching exercise. In this experiment, the denaturation of hen egg-white lysozyme by guanidine hydrochloride is studied. Students perform ratiometric and single-wavelength measurements and obtain thermodynamic parameters for the denaturation process; they also test the reversibility of denaturation. In these studies the advantages of the ratiometric method are highlighted. Students develop analytical skills and, simultaneously, their understanding of the physical-chemical principles behind protein structural changes.