Ghiron C, Bazin M, Santus R
Department of Biochemistry, University of Missouri, Columbia 65211.
J Biochem Biophys Methods. 1988 Apr;15(6):337-48. doi: 10.1016/0165-022x(88)90087-5.
The triplet-triplet absorption spectrum of the sole indole side chain of human serum albumin and its decay kinetics were previously characterized, at room temperature, by using a conventional flash photolysis method [(1978) Proc. Natl. Acad. Sci. USA 75, 1172-1175]. Exploitation of this potentially useful long lived reporter group in protein studies was limited by the excessively large sample size required by that apparatus. The 265 nm laser flash instrument used in the present work avoids this problem at the price of a loss in photo-selectivity. We report that the latter concern can be mitigated. Melittin was studied first because this polypeptide contains a single aromatic residue (W-19), and because its monomeric and tetrameric forms are good models for solvent exposed and buried indole side chains of proteins. For both forms, the indole triplet and neutral radical absorption spectra could be readily time resolved and identified on the basis of shape and differential dioxygen sensitivity. The single tryptophan containing protein human serum albumin was studied next because it contains a large number of other 265 nm absorbing moieties whose transient spectra might complicate the detection of the indole triplet. These transients were shown to not interfere significantly in the wavelength region 450 nm to 600 nm, and, in contrast to the indole triplet, they were relatively dioxygen insensitive. Thus, a facile means is available by which the indole triplet of proteins may be characterized. Subsequently the question of whether this species could be detected in the presence of nuclei acid components was investigated by flashing the phage fd. The putative nucleic acid transients were shown not to interfere and the absorbance of the indole triplet was readily time resolved. The spectral assignment was persuasively confirmed by showing that the indole triplet absorption and phosphorescence emission spectra decay with the same lifetime. The present work thus provides additional evidence for the general applicability of the indole triplet excited state as a long lived intrinsic protein reporter group.
人血清白蛋白唯一吲哚侧链的三重态-三重态吸收光谱及其衰减动力学，先前在室温下通过传统的闪光光解方法进行了表征[（1978年）《美国国家科学院院刊》75卷，1172 - 1175页]。该仪器所需的样本量过大，限制了这种在蛋白质研究中潜在有用的长寿命报告基团的应用。本研究中使用 的265纳米激光闪光仪器以牺牲光选择性为代价避免了这个问题。我们报告称，后一个问题可以得到缓解。首先研究了蜂毒肽，因为这种多肽含有一个单一的芳香族残基（W - 19），并且其单体和四聚体形式是蛋白质中暴露于溶剂和埋藏的吲哚侧链的良好模型。对于这两种形式，吲哚三重态和中性自由基吸收光谱都可以很容易地进行时间分辨，并根据形状和对二氧 化碳的不同敏感性进行识别。接下来研究了含单一色氨酸的蛋白质人血清白蛋白，因为它含有大量其他在265纳米处有吸收的基团，其瞬态光谱可能会使吲哚三重态的检测变得复杂。结果表明，这些瞬态在450纳米至600纳米的波长区域不会产生显著干扰，并且与吲哚三重态不同，它们对二氧化 碳相对不敏感。因此，有一种简便的方法可用于表征蛋白质的吲哚三重态。随后，通过对噬菌体fd进行闪光，研究了在存在核酸成分的情况下是否能检测到这种物质的问题。结果表明，假定的核酸瞬态不会产生干扰，并且吲哚三重态的吸光度很容易进行时间分辨。通过表明吲哚三重态吸收光谱和磷光发射光谱以相同的寿命衰减，有力地证实了光谱归属。因此，本研究为吲哚三重态激发态作为一种长寿命的内在蛋白质报告基团的普遍适用性提供了额外的证据。
