Fluorescence quenching as an indicator for structural fluctuations in liver alcohol dehydrogenase

Abstract

N-Acetyltryptophanamide (NATA), when illuminated anywhere within the 280 nm absorption band, has an emission lifetime of 3.1 ns. The tryptophan residues in liver alcohol dehydrogenase (LADH), however, when excited at 280 nm exhibit 2 lifetimes of .tau.1 = 2.2 and of .tau.2 = 5.7 ns. Excitation at 300 nm yields a single decay of 5.0 ns. At the latter wavelength, only the 2 (equivalent) tryptophan residues buried within the LADH structure are excited. The reaction rate of the NATA fluorescence quenching by ionic and nonionic quenchers is practially independent of the temperature (between 5 and 41.degree. C). The same substances were used to quench the tryptophan fluorescence in LADH. Here (in the same temperature range), the quenching rate decreases drastically with a decrease in temperature. These findings are discussed in terms of conformational fluctuations in LADH, whereby the temporal movement of the polypeptide chains opens channels through which the above quencher molecules can diffuse and reach the tryptophan residues located within the enzyme structure.