HIGH RESOLUTION LASER SPECTROSCOPY OF LANTHANIDE IONS IN BIOCHEMICAL SYSTEMS (EUROPIUM, CALMODULIN, THERMOLYSIN

High resolution site selective laser techniques have been applied to lanthanide ions bound at sites normally occupied by calcium in biochemical systems. Lanthanide ions are functional substitutes for calcium in many biochemical systems. The narrow but weak spectral transitions of the shielded f orbitals of lanthanides can be directly accessed with intense narrowband dye lasers operating in the visible region of the spectrum where few biochemical interferences occur. Although shielded, the f orbitals are slightly affected by the crystal field present at the lanthanide ion site. Different calcium ion sites will consequently affect the 4f('n) to 4f('n) lanthanide transitions differently and this provides the means for site selective laser excitation or fluorescence monitoring. Chapters covering the spectroscopy of biochemical model complexes illustrate the selectivity that can be obtained with high resolution laser techniques.;Spectral separation of europium ions coordinated to different calcium ion sites in the protein thermolysin has been attained and agrees with chemical methods employed to affect the same separation of spectral features. The more recent techniques of fluorescence line narrowing were applied to the europium thermolysin system in the quest for greater site selectivity. Fluorescence line narrowing was observed in the resonant ('7)F(,0) (--->) ('5)D(,0) europium transition but, except for slight narrowing observed in the ('5)D(,0) (--->) ('7)F(,1) transition, no fluorescence line narrowing was observed in the other non-resonant transitions for europium coordinated to thermolysin. This result indicates accidental degeneracies of ions in different sites will limit the resolution enhancement that can be obtained for protein sites binding lanthanide ions.;Site selective laser techniques were applied to the important protein calmodulin. Unique classes of sites were observed for europium bound to calmodulin in both the liquid and solid state. Titrations of calmodulin with europium and terbium were compared. Terbium titrations relied upon energy transfer from a tyrosine amino acid for site selectivity whereas europium titrations, using a narrowband laser for and excitation source, relied upon different crystal field environments for site selectivity. Europium is shown to bind the two high affinity calmodulin sites in a manner analogous to binding observed by magnetic resonance for ('113)Cd('+2).