Torikata T, Forster L S, O'Neal C C, Rupley J A
Biochemistry. 1979 Jan 23;18(2):385-90. doi: 10.1021/bi00569a024.
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.
在pH 7.4的Tris缓冲溶液中记录了脉冲激发后猪心乳酸脱氢酶色氨酸发射的衰减情况。所有色氨酸残基均有发射。通过I(t)-0.53e-t/1.2 + 0.47e-t/68可得到良好的最小二乘双组分拟合。还观察到一个寿命更长的发射体（τ=7.4--8.1纳秒）。结合的NADH强烈淬灭了大部分6.8纳秒的发射，但1.2纳秒的组分相对不受影响。荧光被丙烯酰胺适度淬灭，仅被I-和Cs+轻微淬灭。根据具有1、4和8纳秒三种寿命类别的色氨酸模型讨论了脉冲和稳态荧光。利用酶-NADH复合物的三维结构，根据各个残基的寿命以及对NADH和I-淬灭的敏感性来进行描述。非线性NADH淬灭是由于从Trp-248到NADH的亚基间能量转移。
