Casas-Finet J R, Wilson S H, Karpel R L
Department of Chemistry and Biochemistry, University of Maryland Baltimore County 21228.
Anal Biochem. 1992 Aug 15;205(1):27-35. doi: 10.1016/0003-2697(92)90574-q.
We present an improved procedure for the selective modification of tryptophan residues in proteins. A simple, low-cost set-up allows rapid tryptophan photoreaction upon ultraviolet irradiation in the presence of 2,2,2-trichloroethanol. This photochemical reaction is carried out under native conditions, occurs only in the excited state of tryptophan, and yields a single, as yet unidentified, photoproduct. Except for tyrosine, no reaction with other amino acid side chains are known. Stringent photoselection of tryptophan, ensuring that tyrosine residues are not affected, is achieved in situ without the need for an elaborate system of optical filters or lenses. Illumination with a medium-wave uv lamp of samples placed in disposable, dual pathlength, polystyrene fluorescence cuvettes allows treatment of small sample volumes (greater than or equal to 100 microliters) of various optical density. Chromophore accessibility in oligomeric assemblies or protein-nucleic acid complexes can be assessed by this reaction since the integrity of these structures is preserved. Moreover, this technique can be used to evaluate the involvement of tryptophan residues in catalytic or ligand binding processes.
我们提出了一种改进的方法，用于选择性修饰蛋白质中的色氨酸残基。一种简单、低成本的装置可在2,2,2-三氯乙醇存在下，经紫外线照射实现色氨酸的快速光反应。这种光化学反应在天然条件下进行，仅在色氨酸的激发态发生，并产生一种单一的、尚未鉴定的光产物。除酪氨酸外，已知与其他氨基酸侧链无反应。色氨酸的严格光选择确保酪氨酸残基不受影响，无需复杂的光学滤光片或透镜系统即可原位实现。使用中波紫外灯照射置于一次性、双光程聚苯乙烯荧光比色皿中的样品，可处理各种光密度的小体积样品（大于或等于100微升）。由于这些结构的完整性得以保留，因此可通过该反应评估寡聚体组件或蛋白质-核酸复合物中发色团的可及性。此外，该技术可用于评估色氨酸残基在催化或配体结合过程中的作用。
