Figure 8. Subunit interaction determined by FRET assay. A: Time-dependent changes in the emission spectra of donor and acceptor Alexa Fluor dyes (thiol) that were labeled in αBT162C due to FRET are shown. The emission spectra of αBT162C, excited at 485 nm, were recorded at 5–20 min intervals after mixing an equal amount of donor and acceptor protein at 37 °C. The arrow marks show that donor fluorescence intensity decreases at 519 nm and acceptor emission increases at 565 nm. Emission spectra were recorded for 5 h with the excitation wavelength of 495 nm. B: There was a time-dependent decrease in the emission spectra of thiol-reactive, Alexa Fluor-labeled αBI5C in the presence of Alexa Fluor-labeled αBT162C. Note the absence of Alexa Fluor spectra with 565 nm maximum indicating that there is no energy transfer to the acceptor Alexa Fluor 555 from the donor Alexa Fluor 488. The results suggest that there was no interaction between cysteines in NH₂- and COOH-terminal regions of the αB-crystallin subunits.