Packard B Z, Toptygin D D, Komoriya A, Brand L
OncoImmunin, Inc., College Park, MD 20742, USA.
Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11640-5. doi: 10.1073/pnas.93.21.11640.
Xanthene dyes are known to form dimers with spectral characteristics that have been interpreted in terms of exciton theory. A unique aspect of H-type dimers is the fluorescence quenching that accompanies their formation. Using the principles of exciton theory as a guide, a series of protease substrates was synthesized with a xanthene dye on each side of the cleavage site. To bring the attached dyes into spatial proximity to form a dimer, the molecular design included structure determinant regions in the amino acid sequence. In addition, chromophores were chosen such that changes in absorption spectra indicative of exciton splitting were anticipated. Cleavage of the peptides by a protease resulted in disruption of the dimers and indeed significant absorption spectral changes were observed. Furthermore, substrate cleavage was accompanied by at least an order of magnitude increase in fluorescence intensity. This has allowed determination of intracellular elastase activity using a fluorescence microscope equipped with standard optics.
呫吨染料已知会形成具有光谱特征的二聚体，这些特征已根据激子理论进行了解释。H型二聚体的一个独特之处在于其形成过程中伴随的荧光猝灭。以激子理论原理为指导，合成了一系列蛋白酶底物，在切割位点的每一侧都带有一个呫吨染料。为了使连接的染料在空间上接近以形成二聚体，分子设计在氨基酸序列中包含了结构决定区域。此外，选择发色团时预计会出现指示激子分裂的吸收光谱变化。蛋白酶对肽的切割导致二聚体的破坏，并且确实观察到了显著的吸收光谱变化。此外，底物切割伴随着荧光强度至少一个数量级的增加。这使得能够使用配备标准光学器件的荧光显微镜测定细胞内弹性蛋白酶的活性。
