Schlessinger J, Roche R S, Steinberg I Z
Biochemistry. 1975 Jan 28;14(2):255-62. doi: 10.1021/bi00673a010.
The circular polarization of the luminescence of a chromophore, in addition to its circular dichroism and optical rotatory dispersion, is a manifestation of its asymmetry. In the study of proteins, the circular polarization of luminescence yields more specific information than circular dichroism or optical rotatory dispersion since nonfluorescent chromophores do not contribute, and the spectra of the tyrosine and the tryptophan residues are much better resolved in emission than in absorption. The circular polarization of the fluorescence of the tyrosine and tryptophan residues in derivatives of subtilisin Carlsberg and subtilisin Novo were indeed resolved in this study. The tyrosine residues in the Carlsberg protein, and both tyrosine and tryptophan residues in the Novo protein, were found to be heterogeneous with respect to their optical activity and emission spectra. Changes in the environment of the emitting tyrosine residues in both proteins and in the tryptophan residues in the Novo protein were found on changing the pH from 5.0 to 8.3. The pH dependence of the enzymatic activity of these proteins may thus be due, at least in part, to conformational changes in the molecules. Fluorescence circular polarization also revealed that covalently bound inhibitors at the active site of subtilisin Novo affect the environment of the emitting aromatic side chains, presumably via changes in conformation.
发色团发光的圆偏振，除了其圆二色性和旋光色散外，是其不对称性的一种表现。在蛋白质研究中，发光的圆偏振比圆二色性或旋光色散能产生更具体的信息，因为非荧光发色团不起作用，并且酪氨酸和色氨酸残基的光谱在发射中比在吸收中分辨得更好。在这项研究中，确实解析了嗜热栖热菌蛋白酶和嗜热栖热菌蛋白酶Novo衍生物中酪氨酸和色氨酸残基荧光的圆偏振。发现嗜热栖热菌蛋白酶中的酪氨酸残基以及嗜热栖热菌蛋白酶Novo中的酪氨酸和色氨酸残基在光学活性和发射光谱方面是异质的。当pH从5.0变为8.3时，发现这两种蛋白质中发射荧光的酪氨酸残基以及嗜热栖热菌蛋白酶Novo中色氨酸残基的环境发生了变化。因此，这些蛋白质酶活性的pH依赖性可能至少部分归因于分子中的构象变化。荧光圆偏振还表明，嗜热栖热菌蛋白酶Novo活性位点上的共价结合抑制剂可能通过构象变化影响发射荧光的芳香侧链的环境。
