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  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Journal of fluorescence 4 (1994), S. 187-193 
    ISSN: 1573-4994
    Keywords: Oxygen fluorescence quenching ; tryptophan fluorescence in proteins ; Smoluchowski equation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Physics
    Notes: Abstract The work of Lakowicz and Weber [Biochemistry 12, 4161 (1973)] demonstrated that molecular oxygen is a powerful quencher of tryptophan fluorescence in proteins. Here we report studies of the oxygen quenching of several proteins that have a single, internal tryptophan residue. Among these are apoazurin (Pseudomonas aeruginosa), asparaginase (Escherichia coli), ribonuclease T1 (Aspergillus oryzae), and cod parvalbumin. Both fluorescence intensity and phase lifetime quenching data are reported. By comparison of these data we find that there is a significant degree of apparent static quenching in these proteins. The dynamic quenching rate constants,k q, that we find are low compared to those for tryptophan residues in other proteins. For example, for apoazurin we find an apparentk q of 0.59×109 M −1 s−1 at 25°C. This value is the lowest that has been reported for the oxygen quenching of tryptophan fluorescence.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 0887-3585
    Keywords: cold unfolding ; protein folding ; heat capacity change ; fluorescence ; circular dichroism ; size exclusion chromatography ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Fluorescence and circular dichroism data as a function of temperature were obtained to characterize the unfolding of nuclease A and two of its less stable mutants. These spectroscopic data were obtained with a modified instrument that enables the nearly simultaneous detection of both fluorescence and CD data on the same sample. A global analysis of these multiple datasets yielded an excellent fit of a model that includes a change in the heat capacity change, ΔCp, between the unfolded and native states. This analysis gives a ΔCp of 2.2 kcal/mol/·K for thermal unfolding of the WT protein and 1.3 and 1.8 kcal/mol/K for the two mutants. These ΔCp values are consistent with significant population of the cold unfolded state at ∼0°C. Independent evidence for the existence of a cold unfolded state is the observation of a separately migrating peak in size exclusion chromatography. The new chromatographic peak is seen near 0°C, has a partition coefficient corresponding to a larger hydrodynamic radius, and shows a red-shifted fluorescence spectrum, as compared to the native protein. Data also indicate that the high-temperature unfolded form of mutant nuclease is relatively compact. Size exclusion chromatography shows the high temperature unfolded form to have a hydrodynamic radius that is larger than that for the native form, but smaller than that for the urea or pH-induced unfolded forms. Addition of chemical denaturants to the high-temperature unfolded form causes a further unfolding of the protein, as indicated by an increase in the apparent hydrodynamic radius and a decrease in the rotational correlation time for Trp140 (as determined by fluorescence anisotropy decay measurements). Proteins 28:227-240, 1997 © 1997 Wiley-Liss Inc.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
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