Development of laser techniques for applications in biomolecules and cellular systems.

Abstract

The work presented in this thesis demonstrates how laser flash photolysis techniques can be employed in the application of the study of biomolecules and in cellular environments. The development of a new laser flash photolysis system is described, and a user-oriented, integrated hardware and software solution is presented. The combination of hardware and software used in this approach makes use of the concept of virtual instrumentation through the LabVIEW programming environment. This graphical approach to programming is optimized for data acquisition and instrument control, and for creating a user-friendly environment for the experimenter. Laser techniques have been used to study the biomolecule lignin and some of its model compounds in order to address the issue of light-induced colour reversion of pulp and paper products. High-yield pulps which contain lignin could have new and different industrial uses if the colour could be stabilized. Our work in this field has helped to elucidate the mechanism of yellowing of lignin mainly through the use of lignin model compounds such as $\alpha$-guaiacoxyacetoveratrone ($\alpha$-GAV) and related compounds. Our studies show that the formation of radicals from the excited singlet state is much more important than what is expected from aromatic ketones, and under certain conditions, the singlet pathway is the only degradative one. The results obtained in studies of crystalline $\alpha$-GAV show that efficient excited state deactivation pathways can be created by intermolecular interactions, leading to very good photostability of the molecule. Excited state transients detectable in solution are not present in the crystalline form. In contrast, $\alpha$-(p-methoxyphenoxy)-p-methoxy acetophenone (VII), does produce triplet state absorption on sufficiently long time scale to be detected. This difference is attributed to a different packing structure of crystalline VII which does not allow for efficient excited state deactivation as in the case of $\alpha$-GAV. Pulse radiolysis studies were carried out on VII, giving us insight into the fragmentation of the ketyl radical derived from this molecule. These results demonstrate that this ketyl radical is extremely short-lived and any strategy involving trapping of these types of radicals in the paper environment will most probably prove to be unsuccessful. The properties of the p-methoxyphenacyl radical (IX) were studied and show some interesting aspects of reactivity (acting as a carbon-centred radical), whereas the spectroscopic properties show a long-wavelength absorption which shows some oxygen-centred radical characteristics. These are supported by molecular modeling calculations. This thesis presents the first use of diffuse reflectance laser flash photolysis to directly monitor photochemically generated transients in living cell suspensions. (Abstract shortened by UMI.)