Fluorescence lifetime and anisotropy studies with liver alcohol dehydrogenase and its complexes

• Published in: Biochemistry (Easton) 1986-10, Vol.25 (21), p.6631-6637
• Main Authors: Eftink, Maurice R, Hagaman, Karen A
• Format: Article
• Language: English
• Subjects: Acrylamide; Acrylamides; alcohol dehydrogenase; Alcohol Dehydrogenase - metabolism; Animals; Biological and medical sciences; fluorescence; Fluorescence Polarization; Fundamental and applied biological sciences. Psychology; Horses; Indoles - metabolism; Kinetics; liver; Liver - enzymology; Molecular biophysics; NAD - metabolism; Protein Binding; Spectrometry, Fluorescence; Spectroscopy : techniques and spectras; Tryptophan
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00369a045

From measurements of the apparent phase and modulation fluorescence lifetime of liver alcohol dehydrogenase at multiple modulation frequencies (6, 18, and 30 MHz), the individual lifetimes and fractional intensities of Trp-314 and Trp-15 are calculated. Values of tau 314 = 3.6, tau 15 = 7.3, and f314 = 0.56, at 20 degrees C, are found. These values are in general agreement with values previously reported by Ross et al. [Ross, J.B.A., Schmidt, C.J., & Brand, L. (1981) Biochemistry 20, 4369] using pulse-decay methodology. In ternary complexes formed between the enzyme, NAD+ and either pyrazole or trifluoroethanol, the fluorescence lifetime of Trp-314 is found to be reduced, indicating that the binding of these ligands causes a dynamic quenching of this residue. The lifetime of Trp-314 is decreased more in the trifluoroethanol ternary complex than that with pyrazole. Also, the alkaline quenching transition of alcohol dehydrogenase is found to result in the selective, dynamic quenching of Trp-314. No change in the lifetimes of the two Trp residues is found upon selective removal of the active-site zinc atoms. From studies of the fluorescence anisotropy, r, of the enzyme as a function of added acrylamide (which selectively quenches the surface Trp-15 residue), the steady-state anisotropy of each residue is determined to be r314 = 0.26 and r15 = 0.21. In the ternary complexes the anisotropy of each residue increases slightly.