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

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 (1-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 S1,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. [ABSTRACT FROM AUTHOR]