The commonly used fluorescent probe, fluorescein, can exist in seven prototropic forms. We have used global analysis procedures to reanalyze the absorption data of Diehl and Horchak-Morris (Talanta 34, 739-741, 1987) in terms of five alternative ionization models. We identify the forms of fluorescein present in aqueous solution and the pK a of each ionisation transition. The pKa values of the neutral xanthene, carboxylic acid, and cationic xanthene groups are 6.3, 3.1-3.4, and 3.1-3.4, respectively, and the pKa value of lactonization is 2.4. As a consequence, the neutral form of fluorescein is a mixture of the lactone (70%), zwitterionic (15%), and quinoid (15%) forms. A knowledge of the forms present in solution permits the characterization of their spectral properties. It is shown that the quinoid and monoanion forms have similar absorption spectra, while the zwitterion spectrum is similar to that of the cation but blue-shifted by 3 nm. The emission spectra of the monoanion and quinoid forms are also identified and shown to be similar but not identical. A model for the excited-state reactions of fluorescein is presented.
Human apolipoprotein C-II (apoC-II) self-associates in solution to form aggregates with the characteristics of amyloid including red-green birefringence in the presence of Congo Red under cross-polarized light, increased fluorescence in the presence of thioflavin T, and a fibrous structure when examined by electron microscopy. ApoC-II was expressed and purified from Escherichia coli and rapidly exchanged from 5 M guanidine hydrochloride into 100 mM sodium phosphate, pH 7.4, to a final concentration of 0.3 mg/mL. This apoC-II was initially soluble, eluting as low molecular weight species in gel filtration experiments using Sephadex G-50. Circular dichroism (CD) spectroscopy indicated predominantly unordered structure. Upon incubation for 24 h, apoC-II self-associated into high molecular weight aggregates as indicated by elution in the void volume of a Sephadex G-50 column, by rapid sedimentation in an analytical ultracentrifuge, and by increased light scattering. CD spectroscopy indicated an increase in beta-sheet content, while fluorescence emission spectroscopy of the single tryptophan revealed a blue shift and an increase in maximum intensity, suggesting repositioning of the tryptophan into a less polar environment. Electron microscopy of apoC-II aggregates revealed a novel looped-ribbon morphology (width 12 nm) and several isolated closed loops. Like all of the conserved plasma apolipoproteins, apoC-II contains amphipathic helical regions that account for the increase in alpha-helix content on lipid binding. The increase in beta-structure accompanying apoC-II fibril formation points to an alternative folding pathway and an in vitro system to explore the general tendency of apolipoproteins to form amyloid in vivo.
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