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.

The direct time-resolved fluorescence anisotropy of the single tryptophan residue in the polypeptide hormone adrenocorticotropin-(1-24) (ACTH) and the fluorescence decay kinetics of this residue (Trp-9) are reported. Two rotational correlation times are observed. One, occurring on the subnanosecond time scale, reflects the rotation of the indole ring, and the other, which extends into the nanosecond range, is dominated by the complex motions of the polypeptide chain. The fluorescence lifetimes of the single tryptophan in glucagon (Trp-25) and the 23-26 glucagon peptide were also measured. In all cases the fluorescence kinetics were satisfied by a double-exponential decay law. The fluorescence lifetimes of several tryptophan and indole derivatives and two tryptophan dipeptides were examined in order to interpret the kinetics. In close agreement with the findings of Szabo and Rayner [Szabo, A. G., & Rayner, D. M. (1980) J. Am. Chem. Soc. 102, 554-563], the tryptophan zwitterion exhibits emission wavelength dependent double-exponential decay kinetics. At 320 nm tau 1 = 3.2 ns and tau 2 = 0.8 ns, with alpha 1 = 0.7 and alpha 2 = 0.3. Above 380 nm only the 3.2-ns component is observed. By contrast the neutral derivative N-acetyltryptophanamide has a single exponential decay of 3.0 ns. The multiexponential decay kinetics of the polypeptides are discussed in terms of flexibility of the polypeptide chain and neighboring side-chain interactions.

The difference spectra of binary and ternary complexes of horse liver alcohol dehydrogenase with oxidized and reduced nicotinamide adenine dinucleotides, nicotinamide 1,N6-ethenoadenine dinucleotide, and adenosine diphosphate ribose along with a number of substrate analogues have been measured. These spectra bear a very close resemblance to those obtained by perturbation of the coenzyme(s) and their analogues by acid, NaCl, dioxane, or tert-butyl alcohol. It is inferred that the coenzymes experience a combination of ionic and nonpolar environments at the adenine binding site of the enzyme. This is borne out by published X-ray crystallographic results. The phosphorescence spectra do not indicate the presence of ionized tyrosine in ternary complexes invovling enzyme, coenzyme, and substrate analogues. The ultraviolet spectra can be explained as arising from the perturbation of the coenzyme chromophores upon binding to the enzyme without having to invoke tyrosine ionization.

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