Time-resolved fluorescence of the two tryptophans in horse liver alcohol dehydrogenase

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.

A time-resolved fluorescence study of human copper-zinc superoxide dismutase

“…In our opinion, it seems unreasonable to interpret the two lifetime values obtained for HSOD using the two-exponential model with the presence of two molecular species, one of which accounts for about 10% of the total. At variance with other cases reported and with the classic example of horse liver alcohol dehydrogenase [13,14], the two tryptophauyl residues of HSOD are equivalent. In addition, it is very common to find single-tryptophan proteins with double-or triple-exponential decay [l].…”

Fluorescence‐quenching‐resolved spectra of fluorophores in mixtures and micellar solutions

“…The fitting procedure was performed by an iterative non-linear least-squares method as described previously [15]. The simultaneous analysis of a series of fluorescence quenching data enabled the fluorescence emission spectrum to be resolved into components according to Eqn (5).…”

Fluorescence‐quenching‐resolved spectroscopy of proteins

“…Using single-photon counting techniques and the analysis of fluorescence decay curves as a function of emission wavelengths, Wahl et al [3, 41 first resolved complex fluorescence spectra of two-tryptophan-containing proteins such as Escherichia cofi lac repressor protein [3] and phosphoglycerate kinase [4]. Following a similar method, Ross et al [5] resolved the emission spectrum of alcohol liver dehydrogenase into two tryptophan residues. Using the phase-resolved fluorescence method, it has been shown recently that the spectrum of twotryptophan-containing metalloprotease from Staphylococcus aureus can be decomposed into two components, which have been attributed to each of the tryptophan residues [9].…”