The Investigation of Model Biological Systems Under High Pressure by Fluorescence Spectroscopy

124 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1981.Fluorescence spectroscopy was used to investigate the effect of hydrostatic pressure on three systems: the binding of small ligands to polymers of (beta)-cyclodextrin, the interaction of ethidium bromide with phenylalanine-specific transfer ribonucleic acid from brewers yeast, and the pressure denaturation of lysozyme and chymotrypsinogen as detected by fluorescence polarization.(beta)-cyclodextrin was used as a model for a rigid binding site whose dimensions are unaffected by pressure in the range of one atmosphere to 10 kbar. The complex between poly-(beta)-cyclodextrin and both 1,8-anilinonaphthalene sulfonate and 6-propionyl-2-(dimethylamino)naphthalene is destablized by increasing pressure. The standard volume change upon complex dissociation is -9.3 ml/mole for both ligands. These results are interpreted on the basis of the relative compressibilities of ligand and solvent, and allow calculation of the compressibility of the ligand molecularly dispersed in water.In contrast to the cycldextrins, the binding of ethidium bromide to the high affinity site of transfer ribonucleic acid is accompanied by large changes in the binding site geometry. The standard volume change upon complex dissociation is +25.6 (+OR-) 2.7 ml/mole. Part of this volume change arises from the decrease in volume associated with the stacking of aromatic rings, and part arises from the decrease of the electrostatic repulsion in the negatively charged nucleic acid by the positively charged ethidium.The apparent volume of fluorescein-labelled lysozyme shows a stable, reversible, 60 percent increase when the pressure is raised to 9 kbar calculated from the polarization and fluorescence lifetime under pressure. This can be given a simple interpretation in terms of solvent penetration of the protein structure at high pressures. In contrast, the results with fluorescein labelled chymotrypsinogen are time-dependent and only partially reversible on release of the pressure. They involve conversion to a form with a lower rotational rate at approximately 6 kbar and return to a faster-rotating form at higher pressure. This latter form persists on pressure release.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD