Hagaman K A, Eftink M R
Biophys Chem. 1984 Oct;20(3):201-7. doi: 10.1016/0301-4622(84)87024-6.
The quenching of the fluorescence of liver alcohol dehydrogenase (LADH) by molecular oxygen has been studied by both fluorescence lifetime and intensity measurements. This was done in the presence of 1 M acrylamide which selectively quenches the fluorescence of the surface tryptophan residue, Trp-15, thus allowing us to focus on the quenching of the deeply buried tryptophan, Trp-314, by molecular oxygen. Such studies yielded a Stern-Volmer plot of F0/F with a greater slope than the corresponding tau o/tau plot. This indicates that both dynamic and static quenching of Trp-314 occurs. The temperature dependence of the dynamic quenching of LADH by oxygen was also studied at three temperatures, from which we determined the activation enthalpy for the quenching of Trp-314 to be about 10 kcal/mol. The oxygen quenching of a ternary complex of LADH, NAD+ and trifluoroethanol was also studied. The rate constant for dynamic quenching of Trp-314 by oxygen was found to be approximately the same in the ternary complex as that in the unliganded enzyme.
通过荧光寿命和强度测量研究了分子氧对肝脏乙醇脱氢酶（LADH）荧光的猝灭作用。这是在1 M丙烯酰胺存在的情况下进行的，丙烯酰胺可选择性猝灭表面色氨酸残基Trp-15的荧光，从而使我们能够专注于分子氧对深埋色氨酸Trp-314的猝灭作用。此类研究得到了F0/F的Stern-Volmer图，其斜率大于相应的tau o/tau图。这表明Trp-314发生了动态猝灭和静态猝灭。还在三个温度下研究了氧对LADH动态猝灭的温度依赖性，由此我们确定Trp-314猝灭的活化焓约为10 kcal/mol。还研究了LADH、NAD+和三氟乙醇三元复合物的氧猝灭作用。发现氧对Trp-314动态猝灭的速率常数在三元复合物中与在未结合配体的酶中大致相同。
