Decay-associated fluorescence spectra and the heterogeneous emission of alcohol dehydrogenase.
A procedure is described for using nanosecond time resolved fluorescence decay data to obtain decay-associated fluorescence spectra. It is demonstrated that the individual fluorescence spectra of two or more components in a mixture can be extracted without prior knowledge of their spectral shapes or degree of overlap. The procedure is also of value for eliminating scattered light artifacts in the fluorescence spectra of turbid samples. The method was used to separate the overlapping emission spectra of the two tryptophan residues in horse liver alcohol dehydrogenase. Formation of a ternary complex between the enzyme, NAD+, and pyrazole leads to a decrease in the total tryptophan fluorescence. It is shown that the emission of both tryptophan residues decreases. The buried tryptophan (residue 314) undergoes dynamic quenching with no change in the spectral distribution. Under the same conditions, the fluorescence intensity of tryptophan (residue 15) decreases without a change in decay time but with a red shift of the emission spectrum. There is also a decrease in tryptophan fluorescence intensity when the free enzyme is acid denatured (succinate buffer, pH 4.1). The denatured enzyme retains sufficient structure to provide different microenvironments for different tryptophan residues as reflected by biexponential decay and spectrally shifted emission spectra (revealed by decay association). The value of this technique for studies of microheterogeneity in biological macromolecules is discussed.
D000426:Alcohol Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000818:Animals; D004134:Dimyristoylphosphatidylcholine; D006736:Horses; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008433:Mathematics; D010713:Phosphatidylcholines; D013050:Spectrometry, Fluorescence
D010713:Phosphatidylcholines; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase; D004134:Dimyristoylphosphatidylcholine