The laser in biophysical and analytical chemistry: Investigating biophysical processes and dynamics, as well as analytical techniques, as applied to systems of biological and chemical relevance

Ashby, Kyle
Major Professor
Jacob W. Petrich
Committee Member
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The unique properties of the laser have permitted the investigation of important biological molecules and systems, and aided in the development of the F-Detector, important in the area of food safety. The laser offers narrow pulses of high peak power to probe chromophore interactions with micelles, protein structure and dynamics, excited-state photophysics, and selective fluorescence detection;Using the laser based time-correlated single photon counting technique, the effect of a surfactant on the fluorescence properties of indole, 1-methylindole, and 3-methylindole in aqueous solutions was investigated. 3-methylindole partitions very well into the Brij-35 micelle as indicated by the triple exponential fluorescence decays. An equilibrium constant of 2.6 x 104 M-1 was determined for the 3-methylindole/micelle interaction;Using the same time-resovled technique, tropomyosin mutants containing either tryptophan, 5-hydroxytryptophan, or 7-azatryptophan were expressed in Escherichia coli and their fluorescence properties studied;Utilizing fluorescence upconversion, measurements of hypericin and its methylated analog, O-hexamethoxyhypericin, which possesses no labile protons, confirm excited-state hydrogen atom transfer as the primary photophysical event in hypericin;Laser based pump-probe transient absorption spectroscopy was utilized to determine that the activation energies for excited-state hydrogen atom transfer of hypericin and hypocrellin are respectively, 0.044 +/- 0.008 and 2.12 +/- 0.070 kcal/mol. The low activation energies are consistent with the hydrogen atom transfer reactions being adiabatic;The laser based F-Detector was designed and built for selective detection of fecal and ingesta contamination on meat carcasses as they are processed. F420 was initially utilized as the fluorescent marker, but later investigations revealed the utility of pheophorbide as a marker for fecal contamination. A 100 ms detector time constant offers "instantaneous" detection. Pheophorbide detection limits are as low as 10-10 M;These investigations illustrate the utility and importance of the laser in biophysical and analytical chemistry. The experimental information obtained would otherwise be almost impossible to obtain using more conventional light sources.