Quantitative study of state switching in proteins using a single probe combined with trilinear decomposition

摘要

The present investigation attempts to separate the variation in time domain from steady-state fluorescence and to discuss quantitatively the state-switching of α-chymotrypsin (CHT). The activity of CHT is pH dependent (inactive at low pH, such as 2.2, but active at physiological pH, such as 8). ANS (l-anilinonaphthalene-8-sulfonate) has two states of fluorescence corresponding to different excitation and emission processes. Though a steady-state technique, Excitation-Emission Matrix fluorescence (EEM) can record all the excitation and emission signals for the ANS-CHT complex system. The trilinear decomposition of the constructed three-way data set (using EEM data of different samples) can provide excitation and emission spectra indicating specific excitation and emission processes, respectively, and a quantitative description for the time domain processes. Besides a detailed description of the excitation-emission processes of ANS, the quantitative investigation of state-switching by CHT is also possible because the fluorescence, due to the S_(1,ct) state of ANS, is sensitive to the solvation environment, which is one of the indicators of CHT activity. Finally, the switching output curve of the ANS-CHT system over a wide pH range is obtained. This study proposes a convenient and economical protocol for investigating state-switching in proteins.