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  • Title: Time-resolved fluorescence of the two tryptophans in horse liver alcohol dehydrogenase.
    Author: Ross JB, Schmidt CJ, Brand L.
    Journal: Biochemistry; 1981 Jul 21; 20(15):4369-77. PubMed ID: 7025898.
    Abstract:
    The tryptophan fluorescence decay of horse liver alcohol dehydrogenase, at 10 degrees C in 0.1 M pH 7.4 sodium phosphate buffer, with excitation at 295 nm, is a double exponential with time constants of 3.8 and 7.2 ns. Within experimental error, the two lifetimes remain constant across the emission spectrum. Only the 3.8-ns lifetime is quenched in the NAD+-pyrazole ternary complex, and only the 7.2-ns lifetime is quenched by 0-0.05 M KI. On the basis of these results, we assign the 3.8-ns lifetime to the buried tryptophan, Trp-314, and the 7.2-ns lifetime to the exposed tryptophan, Trp-15. The steady-state lifetime-resolved emission spectrum of Trp-15 has a maximum at approximately 340 nm and that of Trp-15 is at approximately 325 nm. The total time-resolved emission, after 40 ns of decay, has a maximum between 338 and 340 nm and is primarily due to the Trp-15 emission. As a consequence of the wavelength dependence of the preexponential weighting factors, there is an increase in the average lifetime from the blue to the red edge of the emission. This increase reflects the change in the spectral contributions of Trp-15 and Trp-314. Consideration of the spectral overlap between the emission spectra of the two tryptophans and the absorption due to formation of the ternary complex, as well as the distances between the two residues and the bound NAD+, shows that the selective fluorescence quenching in the ternary complex can be accounted for entirely by singlet-single energy transfer. The decay of the fluorescence anisotropy was measured as a function of temperature from 10 to 40 degrees C and is well described by a monoexponential decay law. Over this temperature range the calculated hydrodynamic radius increases from 33.5 to 35.1 A. Evidently, the indole groups of Trp-15 and Trp-314 rotate with the protein as a whole, and there is some expansion of the protein matrix as the ambient temperature is increased.
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