Fluorescence of protonated excited-state forms of 5-hydroxytryptamine (serotonin) and related indoles.
Fluorescence of protonated excited-state forms of 5-hydroxytryptamine (serotonin) and related indoles.
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5-羟色胺（血清素）和相关吲哚的质子化激发态形式的荧光。
DOI：
10.1073/pnas.60.2.598
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发表时间：
1968
影响因子：
11.1
通讯作者：
R. F. Chen
中科院分区：
文献类型：
--
作者：
R. F. Chen
1955 年，Bowman 等人 1 报道，5-羟色胺（血清素）的中性水溶液在紫外线下发出荧光，激发和发射峰值分别为 295 和 330 mjA，而 Udenfriend 等人 2 发现，在强酸性溶液中，5-羟色胺（血清素）在可见光区域发出荧光，峰值发射接近 550 mLA（所有值均未校正）。这种在酸中可见的（绿色）荧光被广泛用于血清素的荧光测定3，但发射物质的性质仍然不清楚。 Udenfriend4 认识到这种绿色荧光的不寻常性质，它在用酸滴定时出现，而吸收或激发光谱没有相应的变化。还注意到，可见光发射涉及异常大的斯托克斯位移，即吸收带和发射带之间的分离。该通讯提供的数据显示，酸中血清素和其他相关化合物的荧光代表了激发态质子化的一个例子。换句话说，吸收光子后，血清素会质子化，而正是质子化形式导致了酸性溶液中的可见荧光。材料和方法。-血清素（Calbiochem）以硫酸肌酐复合物的形式获得，并从水中重结晶。血清素草酸氢 (Calbiochem) 用于一些初步实验，其荧光特性与硫酸肌酐复合物相同。结晶5-羟基色氨酸(Sigma)无需进一步纯化即可使用。 5-甲氧基吲哚-3-乙酸(Sigma)从水中重结晶。 5-甲氧基吲哚（Sigma）从乙醇-水混合物中重结晶。 5-甲氧基色胺盐酸盐(Aldrich)从乙酸乙酯和乙醇的混合物中重结晶。未经进一步纯化而使用的是：5-氟吲哚、3-甲基吲哚、5-甲基吲哚和6-甲氧基吲哚（均来自Sigma）； 4-羟色胺（山德士）；盐酸色胺（Calbiochem）； 5-苄氧基色胺（Aldrich）；和吲哚-N-乙胺（S. Shifrin 博士的礼物）。盐酸奎宁 (Merck) 是一种在薄层色谱上均相的结晶制剂，如前所述。5 盐酸，AR 级 (J. T. Baker)，无需纯化即可使用。氧化氘 (99.9%) 购自纽约州普莱恩维尤的 K and K Laboratories。荧光光谱是用 Aminco-Bowman 分光荧光计获得的，并如前所述对仪器非线性进行校正。6 可见荧光是用发射单色仪中的 R136 光电管和 750-miA 闪耀光栅测量的。通过比较方法7获得荧光量子产率，以 0.1 N H2SO4 中的奎宁为标准，量子产率为 0.54.8 荧光衰减时间通过前面描述的纳秒闪光技术获得。 9 使用与带有 1A2 前置放大器的 Tektronix 556 示波器耦合的荧光衰减时间装置（TRW Instruments, Inc.）。使用装有旋转快门磷光镜附件的 Aminco-Bowman 分光荧光计获得磷光光谱。样品保存在石英毛细管中，悬浮在充满液氮的石英杜瓦瓶中。结果.-从图 1 所示的校正发射光谱可以得出
In 1955, Bowman et al.1 reported that neutral aqueous solutions of 5-hydroxytryptamine (serotonin) fluoresced in the ultraviolet with excitation and emission peaks at 295 and 330 mjA, and Udenfriend et al.2 found that serotonin fluoresced in the visible region with peak emission near 550 mLA (all values uncorrected) in strongly acidic solutions. This visible (green) fluorescence in acid is widely used for the fluorometric assay of serotonin,3 but the nature of the emitting species has remained obscure. Udenfriend4 recognized the unusual nature of this green fluorescence, which appears on titration with acid without a corresponding change in the absorption or excitation spectrum. It was also noted that the visible emission involved an unusually large Stokes' shift, i.e., the separation between absorption and emission bands. This communication presents data showing that the fluorescence of serotonin and other related compounds in acid represents an example of excited-state protonation. In other words, after absorbing a photon, serotonin becomes protonated, and it is the protonated form that is responsible for the visible fluorescence in acid solutions. Materials and Methods.-Serotonin (Calbiochem) was obtained as the creatinine sulfate complex and recrystallized from water. Serotonin hydrogen oxalate (Calbiochem) was used for some preliminary experiments and had fluorescence properties identical to those of the creatinine sulfate complex. Crystalline 5-hydroxytryptophan (Sigma) was used without further purification. 5-Methoxyindole-3-acetic acid (Sigma) was recrystallized from water. 5-Methoxyindole (Sigma) was recrystallized from an ethanol-water mixture. 5-Methoxytryptamine hydrochloride (Aldrich) was recrystallized from a mixture of ethyl acetate and ethanol. Used without further purification were: 5-fluoroindole, 3-methylindole, 5-methylindole, and 6-methoxyindole (all from Sigma); 4-hydroxytryptamine (Sandoz); tryptamine hydrochloride (Calbiochem); 5-benzyloxytryptamine (Aldrich); and indole-N-ethylamine (gift of Dr. S. Shifrin). Quinine hydrochloride (Merck) was a crystalline preparation homogeneous on thin-layer chromatography as described previously.5 Hydrochloric acid, AR-grade (J. T. Baker), was used without purification. Deuterium oxide (99.9%) was obtained from K and K Laboratories, Plainview, New York. Fluorescence spectra were obtained with Aminco-Bowman spectrofluorometers and corrected for instrumental nonlinearity as previously described.6 Visible fluorescence was measured with an R136 phototube and a 750-miA blaze grating in the emission monochromator. Fluorescence quantum yields were obtained by the comparative method7 with quinine in 0.1 N H2S04 as a standard with a quantum yield of 0.54.8 Fluorescence decay times were obtained by the nanosecond flash technique as previously described.9 A fluorescence decay time apparatus (TRW Instruments, Inc.) coupled to a Tektronix 556 oscilloscope with a 1A2 preamplifier was used. Phosphorescence spectra were obtained with an Aminco-Bowman spectrofluorometer fitted with a rotating shutter phosphoroscope attachment. The sample was held in a quartz capillary suspended in a quartz Dewar flask filled with liquid N2. Results.-From the corrected emission spectra shown in Figure 1, it can be