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

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Abstract
The decay of tryptophan emission from pig heart lactate dehydrogenase following pulsed excitation was recorded in Tris buffer solution at pH 7.4. All tryptophan residues emit. A good least-squares 2-component fit is obtained with I(t) = 0.53e-t/1.2 + 0.47e-t/6.8. A longer lived emitter (.tau. = 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 slightly quenched by I- and Cs+. The pulsed and steady-state fluorescence is discussed in terms of a model with 3 lifetime classes of tryptophan, namely 1, 4 and 8 ns. The 3-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.