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

Abstract

The number of applications of fluorescence spectroscopy in different areas of 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.

Exploring the cytotoxic activity of new phenanthroline salicylaldimine Zn(II) complexes

“…The fluorescence emission spectra were recorded at room temperature using the excitation wavelengths of 295, 333, 297, and 338 nm (slits = 5 nm) for phen, amphen, 6 and 7, resulting in emission maxima at 359, 570, 350 and 588 nm, respectively. The fluorescence emission intensities were corrected for the absorption and inner filter effects using UV−Visible absorption data recorded for each sample [48,49]. The fluorescence emission of the samples was recorded from 300 to 660 nm in a 10 mm quartz SUPRASIL® cuvette.…”

“…The fluorescence emission spectra were recorded between 300 and 450 nm by applying an excitation wavelength of 295 nm. UV−visible absorption spectra were recorded for each sample to correct the absorption and inner filter effects [48,49] of the fluorescence emission intensities. Due to the intrinsic emission fluorescence of the quencher (1,10-phenanthroline), the fluorescence emission spectra of the ligand alone, under the same experimental conditions used in the BSA titration assays, were recorded and subtracted to each corresponding emission spectrum.…”

Hands-On Approach to Structure Activity Relationships: The Synthesis, Testing, and Hansch Analysis of a Series of Acetylcholineesterase Inhibitors

“…The advantages of using microplate readers for class experiments have been detailed in a number of publications, 7,8 in particular for the assay of enzyme activity 9,10 and inhibitors. 11 All required calculations to derive final IC 50 values can be easily performed using a basic spreadsheet program such as Microsoft Excel.…”

Integrating bio‐inorganic and analytical chemistry into an undergraduate biochemistry laboratory

“…The educational goals of undergraduate biochemistry laboratories include the exposure to and experience with modern instrumentation , as well as interdisciplinary experience . The literature examples of instrumental analysis in undergraduate biochemistry laboratories have mainly included HPLC , and UV/VIS and fluorescence spectroscopy .…”