Phosphorescence maxima and triplet state lifetimes of NAD+ and epsilon-NAD+ in ternary complexes with horse liver alcohol dehydrogenase.
Phosphorescence maxima and triplet state lifetimes of NAD+ and epsilon-NAD+ in ternary complexes with horse liver alcohol dehydrogenase.
复制标题
NAD 和 epsilon-NAD 在与马肝乙醇脱氢酶的三元复合物中的磷光最大值和三重态寿命。
DOI：
10.1111/j.1751-1097.1989.tb04087.x
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发表时间：
1989
影响因子：
3.3
通讯作者：
Ross,JB
中科院分区：
文献类型：
--
作者：
Rousslang,K;Allen,L;Ross,JB
This paper describes the phosphorescence emission and decay times of NAD and its fluorescent etheno derivative, ε‐NALV+, in the pyrazole ternary complex with horse liver alcohol dehydrogenase (ADH). We show that the ε‐NAD+, triplet state, as well as the tryptophan triplet state, can be utilized to monitor the coenzyme‐enzyme interaction. The decays of NAD+and AMP are single exponential, and the lifetimes are the same within experimental error. The phosphorescence lifetimes, evaluated as single exponentials, are slightly shorter in ε‐NAD4than they are in ε‐AMP. Whereas the decay of ε‐AMP was adequately fit by a single exponential with a time constant of very close to 0.5 s, it was necessary to fit the decay of e‐NAD+to a double exponential. Ternary complexes with NAD+excited at 297 nm exhibit decay kinetics nearly identical to those of ADH by itself. On the other hand, when excitation of the e‐NAD+ternary complex is provided at 313 nm. where there is very little absorption by either tryptophan residue, the decay law of the ternary complex is similar to that of ε‐NAD+in solution. Our results demonstrate that NAD+and ε‐NAD+quench tryptophan phosphorescence in ADH. Normalizing the phosphorescence intensity to the 0–0 vibronic band assigned to Trp‐15 (blue‐edge), we calculate a 21% decrease in the phosphorescence associated with Trp‐314 at stoichiometric saturation of the coenzyme binding sites with NAD‐in the ternary complex. When the active sites are saturated with εS‐NAD+, the relative phosphorescence due to Trp‐314 decreases by 63%. Since ε‐NAD+can be selectively excited in ternary complexes with ADH, it is a more useful probe than NAD+for investigating energy transfer in these complexes.
