Lifetimes and NADH quenching of tryptophan fluorescence in pig heart lactate dehydrogenase

The decay of tryptophan emission from pig heart lactate dehydrogenase following pulsed excitation has been recorded in Tris buffer solution at pH 7.4. All tryptophan residues emit. A good least-squares two-component fit is obtained with I(t)-0.53e-t/1.2 + 0.47e-t/68. A longer lived emitter (r=7.4--8...

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Bibliographic Details
Published in:Biochemistry (Easton) 1979-01, Vol.18 (2), p.385-390
Main Authors: Torikata, Takao, Forster, Leslie S, O'Neal, Clifford C, Rupley, John A
Format: Article
Language:English
Subjects:
Animals
Kinetics
L-Lactate Dehydrogenase
Myocardium - enzymology
NAD
Oxidation-Reduction
Spectrometry, Fluorescence
Swine
Tryptophan
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Summary:The decay of tryptophan emission from pig heart lactate dehydrogenase following pulsed excitation has been recorded in Tris buffer solution at pH 7.4. All tryptophan residues emit. A good least-squares two-component fit is obtained with I(t)-0.53e-t/1.2 + 0.47e-t/68. A longer lived emitter (r=7.4--8.1 ns) is also observed. Bound NADH strongly quenches most of the 6.8-ns emission, but the 1.2-ns component is relatively unaffected. The fluorescence is moderately quenched by acrylamide and only slighty quenched by I- and Cs+. The pulsed and steady-state fluorescence is discussed in terms of a model with three lifetime classes of tryptophan, viz., 1, 4, and 8 ns. The three-dimensional structure of the enzyme--NADH complex is used to develop a description of the individual residues in terms of their lifetimes and sensitivity to NADH and I- quenching. The nonlinear NADH quenching is due to intersubunit energy transfer from Trp-248 to NADH.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi00569a024