Steady-state fluorescence and time-resolved fluorescence monitor changes in tryptophan environment in arginase from Saccharomyces cerevisiae upon removal of catalytic and structural metal ions
| Autor: | Susan M. Green, Jay R. Knutson, Preston Hensley |
|---|---|
| Rok vydání: | 1990 |
| Předmět: |
Manganese
Arginase Chemistry Protein Conformation Metal ions in aqueous solution Inorganic chemistry Fluoroimmunoassay Tryptophan Fluorescence spectrometry Ionic bonding Saccharomyces cerevisiae Photochemistry Biochemistry Fluorescence Protein tertiary structure Metal Structure-Activity Relationship Zinc Apoenzymes visual_art visual_art.visual_art_medium Time-resolved spectroscopy |
| Zdroj: | Biochemistry. 29(39) |
| ISSN: | 0006-2960 |
| Popis: | Yeast arginase is a trimeric protein of identical subunits, each containing three tryptophans. Time-resolved fluorescence and steady-state fluorescence were employed to monitor the effects of removing the weakly bound catalytic Mn2+ as well as the tightly bound structural Zn2+/Mn2+. Resolution of the total native emission spectrum into decay-associated spectra (DAS) yielded components with lifetimes of 0.1, 1.2, and 4.0 ns. Upon removal of the catalytic metal, the intensities increased approximately 20% while the lifetimes increased less than 10%, and the DAS were unchanged except in intensity. The two major components are well resolved, but the 0.1-ns term is small and dominated by scattered excitation. In contrast, removal of the structural metal increased decay times to 0.2, 1.8, and 5.3 ns. More important, both native DAS red-shifted and became indistinguishable. These data suggest that removal of the catalytic metal does little to change the microenvironments of the individual tryptophans while removal of the structural metal causes partial unfolding of the protein. The excitation spectra for the active and inactive trimers were resolved into their excitation DAS (IEDAS), suggesting ground-state heterogeneity of the fluorescent species. In contrast, the excitation spectra of arginase without the structural metal could not be resolved due to the indistinguishable DAS. The tryptophans are quenched by acrylamide but not by cesium or iodide. Global analysis of the acrylamide quenching data resulted in two quenching decay-associated spectra (QDAS) which correlated well with the DAS. Since the apoenzyme does not exhibit tryptophan accessibility to either positive or negative ionic quenchers, one must assume that the "unfolded" monomeric protein retains considerable tertiary structure. |
| Databáze: | OpenAIRE |
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